WO2001035068A1

WO2001035068A1 - Pin and ring type spotting device for sample solution

Info

Publication number: WO2001035068A1
Application number: PCT/JP2000/007908
Authority: WO
Inventors: Junichi Mineno; Haruhisa Sawaragi; Yoshiteru Kora; Minoru Ueda; Masanori Takayama; Kiyozo Asada; Ikunoshin Kato
Original assignee: Takara Bio Inc.
Priority date: 1999-11-10
Filing date: 2000-11-10
Publication date: 2001-05-17
Also published as: KR20020064890A; AU1304901A; CN1390300A

Abstract

A pin and ring type spotting device (1) for sample solution capable of spotting a precious sample solution, without waste, on a substrate, wherein a space (28) is formed between a lower end portion (15) of a vertical arm (4) adjacent to a ring (5) and a lower end portion (26) of a pin (7) opposed to the lower end portion (15) of the vertical arm (4) when the pin (7) is positioned at the lowered position, the space (28) being 0.3 mm or longer in horizontal length or 5 m or longer in vertical length.

Description

Description Spotting device for sample solution by pin & ring method

The present invention relates to a pin-and-ring type sample solution spotting apparatus for spotting a large number of sample solutions, for example, a DNA solution, on a surface of a substrate such as a glass plate. Technology background

As a technique for spotting a large number of DNA solutions on the surface of a substrate such as a glass plate or a membrane filter to produce a DNA chip, a pin & ring type DNA solution spotting apparatus 1 shown in FIG. 4 has been proposed. (GM S4 17 Alayer Genetic 'Microsystems)

As shown in FIG. 4, the spotting apparatus 1 generally includes a solution holding mechanism 2 for holding a predetermined amount of a DNA solution, and a pin mechanism 3 for spotting the solution held in the solution holding mechanism 2 onto a substrate. It is configured. The former solution holding mechanism 2 includes a vertical arm 4 extending in the vertical direction, a ring 5 ′ fixed to the lower end of the vertical arm 4, and a first lifting mechanism 6 for lifting and lowering the vertical arm 4. The ring 5 has, for example, an inner diameter of about 2 mm. The latter pin mechanism 3 is a vertical axis passing through the center of the ring 5.

A pin 7 smaller than the inner diameter of the ring 5, and a pin 7 at a raised position where the pin 7 is retracted above the ring 5 and a pin lower end passing through the ring 5. A second elevating mechanism (8) that moves in relation to a lowered position where the part protrudes below the ring (5).

When spotting a DNA solution on a substrate using the spotting apparatus 1, first, the vertical arm 4 is lowered by the first lifting mechanism 6, and the ring 5 is immersed in the DNA solution 9 (step (a) in FIG. 4). (See (b)). Thereafter, the vertical arm 4 is raised by the first lifting mechanism 6, and the ring 5 is removed from the DNA solution 9 (see step (c) in FIG. 4). With the ring 5 removed from the DNA solution 9, A predetermined amount of the DNA solution 9 is held in the space inside the ring 5 by the surface tension of the DNA solution 9. In the steps (a) to (c) so far, the pin 7 of the pin mechanism 3 has the ring 5 held at the raised position, and the pin 7 is kept out of contact with the DNA solution 9.

Next, the spotting device 1 is moved onto the base 10 (see step (d) in FIG. 4), and the pin 7 is lowered by the second lifting mechanism 8. As a result, the pin 7 penetrates the DNA solution 9 held on the ring 5, and at this time, a small amount of the DNA solution 9 having a substantially constant amount is held at the lower end of the pin 7 (step (e) in FIG. 4). See). Then, the pin 7 is further lowered by the second lifting / lowering mechanism 8 so that the lower end of the pin 7 comes into contact with the base 10, and a small amount of the DNA solution 9 held at the lower end of the pin 7 is transferred to the base 1. (See step (f) in Fig. 4). When the spotting is completed, the pin 7 is raised by the second lifting mechanism 8, returns to the raised position shown in FIG. 4D again, and waits until the same DNA solution 9 is spotted on another substrate.

However, in the spotting apparatus 1 configured as described above, the DNA solution 9 that has entered the gap between the pin 7 and the vertical arm 4 rises by capillary action, and as a result, valuable DNA remains in the internal space of the ring 5. In some cases, the amount of the DNA solution 9 was substantially reduced so that the desired number of spottings could not be performed.

In addition, the decrease in the DNA solution retained in the ring sometimes caused a large difference between the spot size spotted first and the spot size spotted last.

Furthermore, the DNA solution 9 remaining in the vertical arm 4 due to the capillary action may be mixed into another DNA solution held in the ring 5 later. Certainly, the vertical arms 4 etc. are usually washed after the required number of spottings are completed, so not all of the DNA solution that has risen along the vertical arm 4 will be mixed into another DNA solution. In some cases, the washing may be incomplete. In such a case, it is highly likely that the remaining DNA solution is newly added to the ring or mixed into the DNA solution.

Thus, the present invention provides a pin-and-ring type sample solution spotting apparatus in which a precious sample solution held in a ring is not lost by capillary action. The purpose is to provide.

It is another object of the present invention to provide a pin-and-ring type sample solution spotting apparatus that prevents another sample solution from being mixed into a sample solution held in a ring.

In order to achieve such an object, a pin-and-ring type sample solution spotting apparatus according to the present invention is provided with a lower end portion of a vertical arm adjacent to a ring and the vertical arm when the pin is in the lowered position. A vertical gap of at least 0.3 mm or a vertical length of 5 mm is formed between the lower end of the arm and the lower end of the pin facing the lower end of the arm. It is characterized in that the size of the arm and the pin is determined.

In another embodiment of the present invention, a lower end portion of the vertical arm has a small cross section having a small horizontal cross section at a position adjacent to the ring.

In another embodiment of the present invention, the vertical arm and the ring are formed by cutting a tube having the same cross section as the ring, and a lower end of the vertical arm facing a lower end portion of the pin The portion has a plane parallel to a vertical plane that contacts the inner surface of the tube. BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 (a) is a front view of a vertical arm and a ring used in a pin-and-ring type sample solution spotting apparatus according to the present invention.

FIG. 1 (b) is a side view of a vertical arm and a ring used in the sample solution spotting apparatus according to the pin & ring method according to the present invention.

FIG. 1 (c) is an end view of a vertical arm and a ring used in the sample solution spotting apparatus according to the pin & ring method according to the present invention.

FIGS. 1 (d) and 1 (e) are front views of a vertical arm and a ring used in a sample solution spotting apparatus according to the present invention, respectively.

FIG. 2 is a front view of a pin used in a sample solution spotting apparatus using a pin and ring method according to the present invention.

Figures 3 (a) and (b) show the use of the vertical arm and ring of Figure 1 and the pins shown in Figure 2. It is a figure showing a state.

FIGS. 4 (a) to 4 (g) are diagrams illustrating a sample solution spotting apparatus using a pin and ring method and a method of using the same. Embodiment of the Invention

Hereinafter, preferred embodiments of the present invention will be described with reference to the accompanying drawings. The sample solution spotting apparatus of the pin & ring type according to the present invention is characterized by a vertical arm 4 and a ring 5 of a solution holding mechanism 2 and a pin 7 of a pin mechanism 3. It is the same as the spotting device described above. Therefore, in the following description, only the configuration of the vertical arm 4, the ring 5, and the pin 7 will be described, and the overall configuration and operation of the spotting device will not be referred to.

(1) Vertical arm and ring

1 (a) to 1 (e) show enlarged views of the vertical arm 4 of the spotting device. As shown, the vertical arm 4 and the ring 5 are formed by cutting a single straight stainless steel tube 11. The machined vertical arm 4 moves from one end (upper end) to the other end (lower end) with respect to the central axis 12 of the tube 11 (Fig. 1

(from top to bottom of (a) and (b)), the upper end portion 13 having the cross-sectional shape of the tube 11 before processing, and the central axis 1 2 of the tube 11 as shown in FIG. 1 (d). The middle section 14 where most of the cross section of the pipe is cut off at the plane parallel to the pipe, and the cross section of most of the pipe section at the plane parallel to the central axis 12 of the pipe as shown in Fig. 1 (e). It has a lower end portion 15 having a smaller cross section than the intermediate portion 14.

In the present embodiment, as shown in FIG. 1 (d), the cross section of the portion 14 ′ cut off to form the intermediate portion 14 is at least half of the tube cross section. In addition, as shown in FIG. 1 (e), in the cross section of the portion 15 'cut away to form the lower end portion 15, the surface 16 facing the central axis 12 is a tube. It is formed by a plane parallel to the surface that is in contact with the inner surface of 11. However, the surface 16 may be a plane that is in contact with the inner surface of the tube 11 described above (in other words, a plane that includes a tangent to the inner surface of the tube).

The lower end portion 15 has a reduced cross section 17 formed by cutting out a portion adjacent to the ring 5 from both sides. This reduced cross-section 17 is perpendicular to the reduced cross-section 17. Various shapes (for example, a shape in which a notch is formed from only one of the left side and the right side of one of FIG. 1 (a)) can be adopted, provided that the structural strength of the arm 4 does not become less than necessary.

The size of the ring 5 in the direction of the central axis 12 is determined so that a required amount of the sample solution can be held in the internal space.

(2) Pin

FIG. 2 shows an enlarged view of pin 7. As shown in the figure, the pin 7 is formed by processing a rod made of stainless steel, and moves from one end (upper end) to the other end (lower end) with respect to the center axis 20 of the pin 7 (see FIG. 2). From top to bottom), upper end gripper (first part) 2 1, upper base (second part) 2 2, taper part (third part) 2

3, lower base (fourth part) 24, small diameter part (fifth part) 25, extra small diameter part (sixth part) 26, minimum diameter part (seventh part) 27 I have. In addition, the lower end surface of the minimum diameter portion 27 is formed with fine irregularities by blasting using ceramic particles having a diameter of about 30 m so that a predetermined amount of the sample solution can be reliably held on the lower end surface. I have.

(3) Usage condition

As shown in FIGS. 3A and 3B, the vertical arm 4 and the ring 5 configured as described above and the pin 7 are connected to the center axis 12 of the vertical arm 4 and the ring 5 by the center of the pin 7. L, fixed to the first and second elevating mechanisms 6, 8 (see FIG. 4) with the axis 20 aligned. Then, as described above, with the sample solution held in the ring 5, the pins 7 (particularly, the extremely small diameter part 26 and the minimum diameter part 27) are penetrated into the DNA solution in the ring 5, and at this time, the minimum diameter The sample solution held on the lower end face of part 27 is spotted on the substrate.

(4) Vertical arm, ring, and pin dimensions

As shown in Fig. 3, the dimensions of the vertical arm 4 and the pin 7 and especially the size of the lower end 15 of the vertical arm 4 and the extra small diameter part 26 of the pin 7 are as shown in Fig. 3. A space 28 of a predetermined size is formed between the lower end portion 15 and the space 28 so as to prevent the sample solution from rising along the vertical arm in the space 28 due to capillary action. , It is decided. Specifically, the experiment was conducted by changing the size of the space 28 variously. As a result, when the distance (horizontal size of the space 28) between the extremely small diameter portion 26 and the vertical arm 4 opposed thereto was set to 0.3 mm or more, a slight rise in the sample solution due to capillary action was confirmed. However, the rise was slight, and there was no practical problem. Also, when the horizontal size of the space 28 is 0.3 mm and the vertical length of the ultra-small diameter portion 26 (the vertical size of the space 28) is 5 mm or more, the sample solution due to the capillary phenomenon is generated. The rise has further declined. In the most preferable combination, when the distance between the micro-diameter portion 26 and the vertical arm 4 is 0.45 mm or more and the vertical length of the micro-diameter portion 26 is 7.5 mm or more, the sample solution due to the capillary phenomenon is obtained. Was almost completely suppressed. It was also confirmed that the reduced cross-section 17 formed at the lower end 15 of the vertical arm 4 was effective in suppressing the rise of the sample solution due to capillary action. Based on the above, the distance between the very small diameter portion 26 and the vertical arm 4 is 0.3 mm or more, preferably 0.45 mm or more, and the vertical length of the very small diameter portion 26 is 5 mm or more, preferably 7.5. It can be understood that it should be at least mm.

Regarding the preferred embodiment of the vertical arm 4, the ring 5, and the pin 7, the dimensions of each part are shown below.

① Vertical arm and ring (See Fig. 1)

D1: 1.8 mm

D2: 1.2 mm

L1: 5.5 mm

L2: 6. Omm

L c5: 0. Omm

L4: 1.0 mm

W2: 0.6 mm

W3: 0.3 mm

② Pin (See Fig. 2)

And 1: 4.5 mm

L2: 10. Omm

L3: 1. Omm L 4: 6.0 mm

L5: 12.5 mm

6: 7.5 mm

L 7: 1. Omm

01: 2.3 mm

φ 2: 4.Omm

3: 2.3 mm

φ 4: 0.6 mm

5: 0.3 mm

φ 6: 0.20, 0.12, 0.08 mm (Change according to spotting diameter.)

(5) Substrate

The substrate on which the sample solution is spotted using the spotting apparatus of the present invention is not particularly limited as long as it is a material that is practically insoluble, and examples thereof include paper, nylon, cellulose, and nitrocellulose. Plastics and plastic derivatives such as cellulose derivatives, polycarbonate, polystyrene and polypropylene, magnetic or non-magnetic metals, quartz and glass and the like are included. The shape is not particularly limited as long as the sample solution can be spotted by the above-mentioned device, but a plate having a uniform thickness or a substrate on a film is suitable for using the device of the present invention. You.

(6) Sample solution

The sample solution spotted using the spotting apparatus of the present invention is not particularly limited, and may be a solution containing any sample desired to be spotted on a substrate. The device of the present invention is particularly suitable for immobilizing biological materials in the fields of biochemistry, molecular biology and the like. Examples of the sample solution used in the operation include a solution containing a biologically-related material such as a nucleic acid, a protein, a peptide, a carbohydrate, and a drug. The substrate on which such a biological material is immobilized is useful for searching for and detecting a substance having an affinity for the material.

(7) Other

In the above description, the vertical arm, ring, and pin are each made of stainless steel. The pipes and rods were formed by processing, respectively, but the material constituting these is not limited to stainless steel, and may be formed of non-ferrous metals, non-metals including plastics and ceramics.

Further, these are not limited to those formed from integrally formed pipes and rods, and may be formed by assembling separately prepared parts. As is apparent from the above description, the pin-and-ring type sample solution spotting apparatus according to the present invention enables spotless precious sample solutions held in the ring to be spotted on the substrate without use. In addition, the spot size of the sample solution spotted on the substrate has a substantially constant size and shape. Furthermore, since the sample solution does not rise to the vertical arm and remains there due to the capillary action, another DNA solution does not mix with the sample solution held in the ring.

Claims

The scope of the claims

1. a vertical arm (4) extending vertically,

A sample solution holding ring (5) arranged along a horizontal plane and fixed to a lower end of the vertical arm (4);

An ascending position in which the lower end is located along the center axis (1 2) of the ring (5) and parallel to the vertical arm (4) and the lower end is located on the side of the vertical arm (4); (5) has a rod-shaped pin (7) that moves between a lower position where the lower end is located below the ring (5).

Place the sample of the sample solution (9) in the space inside the ring (5).

The pin (7) is passed through the sample of the sample solution (9) held in the gap inside the ring (5) by holding the pin (7) based on the surface tension of (9). The sample of the sample solution (9) is held at the lower end of the pin (7), and the sample of the sample solution (9) held at the lower end of the pin (7) is attached to the surface of the base (10). In the spotting equipment (1) for ringing sample solution,

When the pin (7) is in the lowered position, the lower end (15) of the vertical arm (4) adjacent to the ring (5) and the lower end (15) of the vertical arm (4) ), So as to form a space (28) with a horizontal length of at least 0.3 mm between the lower end portion (26) of the pin (7) and the vertical arm (4). A sample solution spotting device using the pin & ring method, in which the size of the pin (7) is determined.

2. A vertical arm (4) extending vertically,

An ascending position in which the lower end is located along the center axis (1 2) of the ring (5) and parallel to the vertical arm (4) and the lower end is located on the side of the vertical arm (4); (5) has a rod-shaped pin (7) that moves between a lower position and a lower position located below the ring (5) through the lower end.

Place the sample of the sample solution (9) in the space inside the ring (5). The pin (7) is passed through the sample of the sample solution (9) held in the space inside the ring (5) by holding the pin (7) based on the surface tension of (9). A pin and ring, which holds the sample of the sample solution (9) at the lower end and attaches the sample of the sample solution (9) held at the lower end of the pin (7) to the surface of the base (10). In the sample solution spotting device (1),

When the pin (7) is in the lowered position, the lower end (15) of the vertical arm (4) adjacent to the ring (5) and the lower end (15) of the vertical arm (4) ) And the lower end portion (26) of the pin (7), the vertical arm (4) and the pin (6) are formed so as to form a space (28) with a vertical length of 5 mm or more. 7) A pin-and-ring type sample solution spotting device characterized in that the size of the sample solution is determined.

3. The lower end of the vertical arm (4) has a small cross section (17) with a small cross section at a position adjacent to the ring (5). A sample solution spotting device according to the pin & ring method described in Crab.

4. The vertical arm (4) and the ring (5) are formed by cutting a pipe (1 1) having the same cross section as the ring (5), and the lower end of the pin (7) is formed. The lower end portion (15) of the vertical arm (4) facing the section (26) has a plane (16) parallel to a vertical surface in contact with the inner surface of the pipe (11). 4. The sample solution spotting apparatus according to any one of claims 1 to 3, wherein the sample solution is spotted by a pin and ring method.

5. The pin-and-ring type sample solution spotting apparatus according to any one of claims 1 to 4, wherein the sample is DNA.