JP2001074745A - Biochip preparation method and biochip-preparing device using it - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method and device for preparing a biochip that does not entwine a molecule, and does not cover a region required for hybridization. SOLUTION: In the biochip preparation method where a DNA, an RNA, or a protein cell 3 is prepared in an array shape on the surface of a substrate, the DNA, the RNA, or the protein chip is prepared by either one or both of a process for allowing solution containing the DNA, RNA, or protein to adhere from the lower part of the substrate, and a process for applying plus and minus voltages from both the sides of the substrate and solution adhering to a substrate 2.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to DNA, RNA,
The present invention relates to a so-called biochip manufacturing method and an improvement of an apparatus for manufacturing cells such as proteins in an array on a substrate.

[0002]

2. Description of the Related Art Here, a DNA chip will be described as an example. DNA chips are generally 1-10 cm
^{It is} a size of ^{2 and is} an array of thousands to hundreds of thousands of DNAs in this region. As a method for producing a DNA chip,
Conventionally, a method of attaching a cDNA fragment prepared by a polymerase chain reaction (PCR) or the like to a substrate such as a slide glass or silicon using an arrayer pin is well known.

In this method, as shown in the principle diagram of FIG. 3, the tip of a pin 1 is immersed in a washing solution in a washing solution tank 2 and washed, and then the pin 1 is moved. A pin is immersed in the inside to attach DNA to the tip of the pin. Subsequently, the pin 1 is moved and stamped on the slide glass 4 to produce a DNA chip.

[0004]

However, such a method has the following problems. Since the solution is simply deposited on the slide glass 4 and allowed to dry naturally, long molecules are entangled as shown in FIG. 4 or extend laterally to cover the region 5 required for hybridization. There is. One of the problems is that the entanglement or overwrap phenomenon is unstable.

The term “hybridization” refers to a method in which a single-stranded denatured nucleic acid is hybridized with another single-stranded nucleic acid containing a complementary base sequence through a sequence-specific hydrogen bond under appropriate conditions. It is well known as an analysis method utilizing formation. Specifically, using a nucleic acid having a known sequence as a probe, a sample is examined for a complementary sequence to be a target. At this time, by attaching a label to the hybrid formed by the probe and the target, it is possible to detect and quantify the complementary sequence in the sample.

SUMMARY OF THE INVENTION An object of the present invention is to solve the above-mentioned problems, and to provide a biochip manufacturing method capable of easily manufacturing a biochip which does not cause entanglement of molecules and does not cover a region required for hybridization. And a biochip manufacturing apparatus using the same.

[0007]

In order to achieve the above object, the present invention provides a method for producing a biochip in which cells of DNA or RNA or protein are produced in an array on a substrate surface, the method comprising the steps of: Attaching a solution containing DNA or RNA or protein from the above, said substrate side,
The DN is applied by one or both of the steps of applying positive and negative voltages from the solution side attached to the substrate.
A or RNA or protein chips are produced.

[0008] According to such a method, it is possible to prevent the molecules attached to the substrate from being entangled, and to produce a biochip in which the molecules are suspended from the substrate.

[0009]

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS The present invention will be described below in detail with reference to the drawings. First, the principle of the present invention will be described. As shown in FIG. 1A, the pin 1 to which the solution containing DNA is attached is stamped from below the slide glass 4. When you release the pin,
Due to gravity, the liquid hangs down from the slide glass 4 as shown in FIG. The liquid form stabilizes.

Next, as shown in FIG. 1B, electrodes 16 and 17 are placed on the side opposite to the slide glass 4 side, and a voltage is applied to apply a negative charge to the slide glass 4 side and a positive charge to the opposite side. give. Since DNA is always negatively charged, the DNA in the solution extends toward the positive electrode as in DNA electrophoresis.

By drying as it is,
As shown in FIG. 1 (c), the DNA was immobilized and did not cover DNA entanglement or the hybridization region.
A tip in which the NA molecule extends (hangs down) is formed.

FIG. 2 is a main part configuration diagram showing one embodiment of a chip manufacturing apparatus according to the present invention. A washing liquid part 11, a solution part 12 containing DNA, and a holding part 13 holding the slide glass 4 are arranged on the ceiling of the frame 10. A pin support 14 that supports the pins 1 is mounted on a stage 15 provided on the floor of the frame 10 so as to be movable in the lateral direction. The pin support 14 can move the pin 1 up and down as appropriate.

For the cleaning liquid section 11 and the solution section 12, for example, a sponge-like medium or a means utilizing surface tension is used so that the respective liquids do not drip.

Electrodes 17 and 16 are provided on the upper surface of the slide glass 4 and below the slide glass 4, respectively, and a voltage is appropriately applied between the electrodes. Further, a drying unit (not shown) for drying the DNA solution attached to the slide glass 4 is provided near the slide glass 4 as necessary.

The drive / control means for moving the pin support 14 on the stage 15 and the vertical movement of the pin 1, the means for applying voltage to the electrodes, and the means for driving the drying means are realized by using known means. it can. The description of these well-known means is omitted here.

The operation in such a configuration will be described.
The pin 1 is lowered, and the pin support 14 is moved directly below the cleaning liquid unit 11. Lift the pin 1 and clean the pin tip with the cleaning solution 1
Poke into 1 and wash. Next, the pin is lowered, and the pin support 14 is moved directly below the solution section 12, where the pin 1 is raised and brought into contact with the solution section 12 to cause the solution to adhere to the pin tip.

After the solution is attached to the tip of the pin, the pin is lowered, and the pin support 14 is moved to just below the slide glass 4. The pin 1 is pushed up to stamp the lower surface of the slide glass 4. After stamping, lower the pin tip away from the liquid.

Next, a voltage is applied to the electrodes 16 and 17 to
NA is extended in the positive electrode 16 direction. In that state, the drying means is operated to dry. The DNA is attached to the slide glass 4 as shown in FIG.

The present invention is not limited to the above embodiment. For example, the number of pins is not limited to one, and a pin array in which a plurality of pins are arranged may be used. In addition, the cleaning liquid unit 11 and the solution unit 12 are not disposed on the ceiling of the frame,
It may be arranged on the floor of the frame 10. Alternatively, a washing tank or a solution tank (both not shown) may be used as in the conventional case.
In such a case, it is necessary to perform the cleaning and the solution adhesion by rotating the inverted pin forward (downward).

Further, both the inversion and the charging are not necessarily required, but only one of them may be performed. In addition, DNA was stamped on the slide glass surface with pins, but DN
A may be attached.

Furthermore, the voltage applied to the electrodes is positive
The minus may be switched as appropriate. DNA can be loosened by such switching.

The present invention is not limited to DNA, but may be RNA or protein.

[0023]

As described above, according to the present invention, a biochip in a state in which molecules attached to a substrate are prevented from being entangled and molecules hang down from the substrate and do not cover a region necessary for hybridization can be easily obtained. Can be created.

[Brief description of the drawings]

FIG. 1 is a diagram illustrating the principle of the present invention.

FIG. 2 is a configuration diagram showing one embodiment of an apparatus according to the present invention.

FIG. 3 is a diagram for explaining the principle of a conventional chip making method.

FIG. 4 is a diagram for explaining entanglement of molecules on a substrate.

[Explanation of symbols]

 DESCRIPTION OF SYMBOLS 1 Pin 2 Substrate 3 Cell 11 Cleaning liquid part 12 Solution part 13 Holding part 14 Pin support 15 Stage 16, 17 Electrode

Claims (4)

[Claims] 1. A method for preparing a biochip in which cells of DNA, RNA or protein are prepared in an array on a substrate surface, the method comprising: attaching a solution containing DNA, RNA or protein from below the substrate; A method for producing a DNA or RNA or protein chip by one or both of the steps of applying positive and negative voltages from the solution side attached to the substrate and the solution side attached to the substrate. . 2. A biochip production apparatus for producing DNA or RNA or protein cells in an array on a substrate surface, comprising: means for attaching DNA, RNA or protein to the substrate from below; A biochip manufacturing apparatus, comprising: one or both of voltage applying means for applying positive and negative voltages from a solution side attached to a substrate. 3. The biochip manufacturing apparatus according to claim 2, wherein the means for attaching the solution is configured to attach the solution to the substrate surface using a pin or a pin array. 4. The means for adhering the solution is similar to the method of adhering the solution to the substrate surface when washing the pin tip and when adhering a solution containing DNA, RNA or protein to the pin tip. The biochip manufacturing apparatus according to claim 3, wherein the cleaning liquid and the solution are attached by contacting from below or conversely from above.