KR100340126B1 - Automated Apparatus for Microarraying Biological Samples - Google Patents

Abstract

The present invention provides an automatic sample microarray apparatus for finely arranging biological samples such as nucleotides and proteins on a substrate by capillary tubes 1 for taking biological samples in the plate 2 and spotting them on the substrate. ; The capillary includes a tip, wherein the inner diameter of the capillary is 0.5 to 3 mm, the length of the tip portion is 2 to 20 mm, the inner diameter of the tip end is 50 to 300 μm, and the capillary is X A driving source including a motor for moving in the Y, Z-axis direction; The rack 2 has a structure that can be stacked in the vertical direction and the circumferential direction, the rack 10 is rotatably operated up and down and removable; A withdrawal hook 13 for pulling out a plate from the rack in a horizontal direction; a fixing plate 18 on which a vacuum pad for fixing a substrate is mounted; Cover opening and closing means (14) for opening the cover of the plate: and an automatic sample microarray apparatus for spotting and arranging a biological sample on a substrate, characterized in that it comprises a washing block for cleaning the capillary . According to the present invention, micro-arranged microchips having biomolecules can be easily manufactured in large quantities by spotting tens of thousands of biological samples at a high density in a fixed amount within 1 cm 2 on a solid plane without operator intervention. .

Description

Automated Apparatus for Microarraying Biological Samples

The present invention relates to an apparatus for finely arranging biological samples, and more particularly, to an automated biological sample microarray apparatus for finely arranging biological molecules such as nucleotides, proteins, etc. on a solid substrate.

As the human genome project continues, a large amount of genome information has been revealed continuously, and the development of technology for efficiently interpreting the function of all genes of various organisms based on the already known genome sequences is required. As a post-genome research subject, terms such as functional genomics or system biology are frequently used. These are the ways in which we can use the results of genome projects to make them useful. Its typical method is a DNA chip technology in which a plurality of DNA molecules are arranged in a sequence such as slide glass or silicon.

DNA chip, also known as DNA microarray, is a very useful technique for simultaneous interpretation of gene expression, mutations, and polymorphisms, and it is necessary for observing the expression level of known genes in large quantities or for discovering new genes. Lipshutz, RJ et al. (1995) Biotechniques 19, 442-447; Chee, M. et al. (1996) Science 274, 610-614. The basic concept of the DNA chip is a method of reverse hybridization on a substrate. The hybridization method of probing DNA fragments separated by size by electrophoresis into DNA has been used as a standard tool for genetic analysis for more than 20 years. Hybridization kinetics is complicated because it is impossible to experiment with different kinds of probes at the same time, and because a solvent-permeable substrate is used. If the probe is immobilized on the matrix, labeled messenger pools or variable regions on the gene can be used to easily identify the proportion of individual transcripts or polymorphisms / mutants present in the sample. Will be. This is reverse hybridization, which has been utilized as a dot blot or a slot blot using a filter. In short, to determine polymorphism in a specific gene, all kinds of oligomers that can be specifically bound to each allele are fixed in a filter, and then the amplification and labeling of the gene under test is carried out by PCR. This is to check which oligomers are hybridized with the filter. In this case, the use of an impermeable support makes the hybridization reaction very simple, and it is much more advantageous in terms of time and economy since it can be simply performed by covering a cover glass with only a few microliters of solution.

In the 90's, a variety of techniques for manufacturing DNA chips with high-density bases were proposed. Such high density DNA chip placement methods can be broadly classified into four types: spotting (or contact printing) method, non-contact printing method, and photolithography method.

As a spotting method, recently, a pin needle such as a pointed needle is immersed in the sample solution, and then a small amount of the solution is taken by sequentially moving the slide glass arranged on the work table. In the case of needle-type solid pins, samples are buried every time spotting is used in some arrayers or colony picking robots. In addition, a method was used to precisely machine the pin tip by wire cutting to create a groove of 25 microns width with the shape of a fountain pen nib (DeRisi, JL et al. (1997) Science 270). 680-686), this type of pin sucks a few microliters of solution once soaked into the sample by capillary action, allowing spotting on multiple slides, but creating a groove in the center of the manufacturing process. It is very difficult, the amount of solution to be taken can not be arbitrarily controlled, and a number of problems may arise, such as abrasion may occur when contacted on a solid substrate.

Non-contact printing is a method of actively controlling the amount of solution by using heat, solenoid actuators, or piezoelectric elements. In general microarray applications, there are many types of samples to be taken. If the sample is extracted from the sample reservoir, a separate container must be prepared for each sample, and there is a problem in that it is very inconvenient to use due to the dead capacity inside the channel.

Photolithography is the most recent technique for making DNA chips, and photolithography is one of the original semiconductor fabrication processes (Affymetrix, CA). This method of synthesizing oligomers directly on a chip using protecting groups which are separated by UV can in principle produce oligomers of the desired combination and can be synthesized in parallel (Pease, AC et al. (1994) Proc. Natl.Acad.Sci. USA 91, 5022-5026). First, a synthetic linker having protecting groups that are eliminated by UV is evenly placed on the glass plate, and the UV light passing through the area activates a certain area of the linker on the glass plate since the parts where the synthesis is desired to be started are drilled in several places. The method below is similar to solid phase oligonucleotide synthesis. The first hydrozyl-protected nucleotide is reacted with this surface to fix one type of base at the desired location. Four photomasks are needed to attach one layer of base to every position on the glass plate, so if you want to produce a 25-mer probe array, you need a 4x25 = 100 photomask. However, this method is a high-level technology that is not readily available to ordinary researchers.

As described above, methods for manufacturing biomolecule microchips are very difficult and difficult for ordinary researchers to use, and when the conventional spotting method is used, since the material of the solid fin is sus, Abrasion is a problem, and the quill method has also been limited to dozens of spots, and when other capillaries are used, spots do not form well. Therefore, improvement of pins to form spots is urgently needed. And, there was a need for an apparatus capable of finely arranging samples by spotting automatically and easily and simply.

Accordingly, the present inventor has made intensive studies in order to spot a sample on a solid substrate to make microarrays, thereby producing a capillary tube which can form fine spots efficiently, and precisely a plurality of capillary tubes on the X, Y, and Z axes. The invention completes the invention knowing that a high density spot can be formed on a solid substrate by automatically spotting and finely arranging a large sample by adopting a movable driving source and a rack structure for storing a large amount of plates. Was done.

Accordingly, an object of the present invention is to provide an apparatus for automatically microarranging biological samples for automatically spotting biological samples on a substrate to form microspots and microarraying spots on the substrate. will be.

1 is a schematic view of the capillary of the present invention.

2 is a plan view of an automatic sample microarray apparatus of the present invention.

3 is a front view of FIG. 2;

4A is a side view of FIG. 2;

4B is a partial side view of FIG. 2.

<Description of the symbols for the main parts of the drawings>

1: capillary 2: plate

10: rack 13: plate withdrawal hook

18: ultrasonic cleaning block 19: fixed plate

The present invention provides an apparatus for finely spotting and arranging a biological sample on a substrate, comprising: a capillary tube (1) for taking a biological sample in a plate (2) and depositing it on a substrate; The capillary includes a tip portion, wherein the capillary inner diameter is 0.5 to 3 mm, the length of the tip portion is 2 to 20 mm, and the inner diameter of the tip end is 50 to 300 μm. A drive source including a motor for moving the motor in the X, Y, and Z axis directions; The rack 2 has a structure capable of stacking the plate 2 in the vertical and circumferential directions, and the rack 10 is movable up and down and rotatable and detachable; A withdrawal hook 13 for pulling out a plate from the rack in a horizontal direction; a fixing plate 19 on which a vacuum pad 20 for fixing a substrate is mounted; Cover opening and closing means (14) for opening the cover of the plate: and automatic sample micro-array arrangement for arranging by sequentially spotting the biological sample on the substrate, characterized in that it comprises a washing block for cleaning the capillary to provide.

The material of the capillary is preferably selected from the group consisting of soda lime, Borosilicate, Quartz.

The cover opening and closing means 14 is characterized in that the vacuum pad 20 is mounted.

The cleaning means is characterized in that it comprises an ultrasonic cleaning block (18).

As described above, according to the apparatus of the present invention, tens of thousands of samples are spotted on a substrate at a constant capacity and at regular intervals within 1 cm 2 of a solid plane to form microspots, and the microspots are densely arranged to be biologically formed on the substrate. Biomolecule microchips with a high density of samples can be fabricated.

Hereinafter, preferred embodiments of the present invention will be described in detail with reference to the drawings, but the present invention is not limited thereto.

The present invention relates to an apparatus for finely arranging a sample on a substrate by spotting, and the sample may be microarrayed by forming spots at regular intervals on the substrate.

Here, the sample refers to a solution containing biomolecules such as DNA, RNA, protein, etc., including oligonucleotides as a substance to be analyzed. In an embodiment of the present invention, the samples may be spotted at regular intervals on the substrate with a spot diameter of about 100 μm, so that biomolecule microchips in which biomolecules are densely integrated may be manufactured.

In the present invention, the substrate may be a solid substrate. For example, a glass plate, a nylon film, a silicon film, or the like may be used, and commercially available slide glass may be used. The multi well plate used in the present invention can be modified in various ways. Commonly used 96 well plates, 384 well plates and the like can be preferably used.

Referring to the capillary tube of the present invention with reference to Figure 1, the inner diameter of the capillary tube 1 is 0.5 to 3 mm, preferably 0.5 mm to 2 mm, the capillary tube includes a tip, the length of the tip portion It is 2-30 mm, Preferably it is 2-20 mm, and the inner diameter of a tip end is 50 micrometers-300 micrometers. In the formation of the capillary tube of the present invention, the coil is wound around the glass tube, heated, and then a tip portion is formed by using a puller. The capillary is made of glass, which can be selected from soda lime, borosilicate, and quartz. Since the capillary is made of glass, it has less abrasion when friction with the substrate and increases stability when performing spotting. In the present invention, the shape of the tip portion of the capillary is characterized in that it differs from the conventional oblique form to give stability. The capillary tube of the present invention can be used repeatedly for hundreds or thousands of spottings at a time. In the present invention, the capillary tube 1 can be formed in multiple numbers, thereby saving time by allowing multiple spots to be formed at the same time. 2, the weight 24 is inserted into the upper portion of each capillary, and then the capillary is inserted into the square fixing jig 25 by weight, and then the fixing jig ( In order to move 26), a spot is formed by attaching a shaft, connecting a ball screw and a stepping motor, and repeating the capillary tube 8 on and off the substrate by a motor. Here, the weight 24 serves to help the gravity action, anti-rotation and positioning of the capillary tube. In the present invention, the spotting can be performed using four 32 vertically and four vertically eight capillaries, but the number of capillaries used can be appropriately adjusted according to the number of spots to be made and the plate used. The capillary tube may be used for spotting several times at one location, or may be moved to another location to repeat the same operation. When the capillary tube 1 containing the sample is brought into contact with the substrate, the sample forms spots on the substrate, and spots are similarly formed on other substrates.

Since the inner diameter of the capillary tip determines the diameter of the spot, the inner diameter of the capillary tip can be adjusted in various ways. The diameter of the spot formed when the inner diameter of the capillary tip is 100 μm corresponds to this. In the present invention, a capillary tube having a uniform inner diameter of the tip is used to make the size of the spot formed uniformly. The spacing between the spots is adjusted according to the number of spots to be formed on the substrate so that the spots do not overlap. In the present invention, the sample can be uniformly microarrayed on the solid substrate in a short time using a 96 well plate or a 384 well plate.

In addition, when the present invention is described with reference to FIGS. 2 to 4, the rack 10 of the present invention has a plate 2 in which 12 plates are stacked up and down, and a columnar shape in which six plates can be stacked in a column shape. It has a structure in which a total of 72 (12x6) pieces are stacked in steps. This rack structure has the advantage of being able to process 27,648 (384x72) samples in one operation when using a 384 well plate to process a large amount of samples. The rack 10 on which the plates 2 are stacked is automatically moved by a ball screw, a timing belt connected to it, and a stepping motor, and rotatably moved by a stepping motor 11 and a belt 12 so that the desired plate can be moved. Can be selected from the rack. The rack of the present invention is detachable so that it can be easily removed from the device to mount a plate, and can be mounted back to the device. From the rack 10, the plate 2 is withdrawn by the plate withdrawal hook 13, and the sample in the plate 2 is taken by the capillary tube after the cover is opened by the cover opening / closing means 14 (Fig. 3). A vacuum pad 15 is mounted on the cover opening and closing means, and the plate cover is attached to the vacuum pad 15 by the vacuum suction action of the vacuum pump. After all the samples in the plate are used, the cover of the plate 2 is automatically closed by the cover opening and closing means 14, and then moved back to the rack, and the operation of taking the next plate proceeds in the same manner. The plate withdrawal hook and lid opening and closing means are both moved by a drive source including a timing belt and a stepping motor. Automatic removal of the plate is possible by the operation of the rack, plate withdrawal hook and cover opening and closing means.

As shown in FIG. 2, the fixed plate 19 is equipped with a vacuum pad 20, on which a substrate is placed. The vacuum pad is connected to the vacuum pump so that the substrate is not disturbed or moved during operation. Moreover, the groove | channel 22 is formed in the circumference | surroundings on which a board | substrate is placed in a fixed plate. The vacuum pad is controlled whether or not the vacuum by the opening and closing of the control valve 21. The fixing plate 19 may be arranged as many substrates as desired. Placing the substrate on the stationary plate in the conventional manner causes the substrate to move even with the operation or minute movement of the device, making it impossible to create a desired spot. In the present invention, the vacuum pad is attached to the fixed plate so that the substrate is in close contact with the fixed plate so that the substrate does not move at all.

In the present invention, in order to move the capillary in the X-axis, Y-axis, Z-axis direction, the LM guide 4 is installed on the X-axis, and the ball screw 3 is installed between the two LM guides, and then Stepping motor (5) is connected to enable the precise movement on the X-axis, and to be moved up and down by the ball screw (6), ball bush or LM guide and stepping motor (6) in the Y-axis direction, It is as above-mentioned in a Z-axis direction. In the present invention, the moving speed to the X-axis, the Y-axis, and the Z-axis is controlled by the control means, so that the moving speed of the capillary to the X, Y, and Z axes can be controlled.

As shown in FIG. 2, the present invention includes a means for cleaning a capillary, the cleaning means comprising a cleaning block 16 containing a cleaning liquid, a vacuum block 17 for removing a sample by vacuum and an ultrasonic cleaning. Block 18. After the capillary tube is washed in the washing block, the residual solution is removed by vacuum in the vacuum block. The washing block is formed with an inlet through which the washing liquid is supplied by the pump and a drain hole through which the washing liquid is not overflowed. After washing with the washing solution, a vacuum block was included to remove the sample by vacuum, and a vacuum tube was formed at the bottom. During vacuum washing, a fixing jig including a capillary tube is inserted into the vacuum block to efficiently discharge the solution in the capillary tube by depressurization. The ultrasonic cleaning block has a vibrator installed to increase the cleaning effect by ultrasonic waves. After taking a sample and spotting, the remaining sample is immersed in the washing block 16, washed, and then put in the vacuum block 17 to further remove the sample in the capillary by vacuum, followed by ultrasonic cleaning, Take a new sample again. Therefore, different types of samples can be spotted repeatedly.

In the present invention, looking at the operation sequence, when the switch is raised, the initialization proceeds, and when the rack 10 is moved up and down or rotated by the motor, the plate 2 is removed and moved by the plate withdrawal hook 13 and the cover The cover is automatically opened by the opening and closing means 14, and then the capillary tube 1 takes a sample in the plate 2. Next, the capillary containing the sample is moved to a desired position on the X axis and the Y axis, and then moved downward to close the end of the capillary 1 tip on the substrate and to form a spot. After the spot is formed, the capillary is moved to the next position and forms a spot in the same position on another substrate. In this way, the spotting is repeatedly performed as many times as desired at the desired position on the substrate by the capillary tube. If you spot the 100 substrates, the capillary buried with the sample will be spotted 100 times. After spattering as many times as desired, the remaining capillary sample is optionally immersed in the washing block 16 and the ultrasonic washing block 18 to perform the cleaning step by step, and finally by vacuum suction in the vacuum block 17. Remove the sample completely. After the cleaning process, the capillary takes a new sample and moves back onto the substrate for the same spotting. Through this process, microchips in which biological sample spots are finely arranged may be manufactured. All the above processes are automatically controlled by control means, for example a program. By using the automatic sample microarray apparatus of the present invention, a large amount of samples of tens of thousands or more can be processed in a single operation, and since the operation is very simple and easy, the biological sample is microarrayed on a solid substrate without user intervention. Microchips can be manufactured in large quantities.

In the present invention, the distance between the spot and the spot by the control means is adjusted in units of at least 5㎛, the spotting speed is able to form seven spots per second, the movement amount of the X-axis, Y-axis moves to at least 5㎛ To provide excellent position accuracy and position reproducibility.

As described above, by using the automated sample microarray apparatus of the present invention, microchips in which biomolecules are microarrayed by spotting and microarraying tens of thousands of desired biological samples in a predetermined amount within 1 cm 2 on a solid plane are used. It can be produced quickly and easily in large quantities without any intervention.

Claims (4)

An apparatus for finely spotting and arranging a biological sample on a substrate, the apparatus comprising: a capillary tube 1 for taking a biological sample in a multi-well plate and depositing the biological sample on a substrate; the capillary tube includes a tip, the inner diameter of the capillary tube Is 0.5 to 3 mm, the length of the tip is 2 to 20 mm, the inner diameter of the tip end is 50 to 300 ㎛, drive source including a motor for moving the capillary in the X, Y, Z axis direction and; The multi well plate (2) has a structure that can be stacked in the vertical direction and the circumferential direction, the rack (10) that is movable up and down and rotatable and detachable; A take-out hook 13 for pulling out a plate from the rack in a horizontal direction: a fixing plate 19 on which a vacuum pad 20 for fixing a substrate is mounted; Cover opening and closing means (14) for opening the cover of the plate: and automatic sample micro-array apparatus for spotting and arranging a biological sample on the substrate, characterized in that it comprises a washing block for washing the capillary. The automatic sample microarray apparatus of claim 1, wherein the capillary material is selected from the group consisting of Soda lime, Borosilicate, and Quartz. The automatic sample microarray apparatus according to claim 1, wherein the cover opening / closing means (14) is equipped with a vacuum pad (20). 2. The apparatus of claim 1, wherein the cleaning block comprises an ultrasonic cleaning block (18).