JP2003083965A - Protein/nucleic acid analyzing chip - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide an efficient and economical protein/nucleic acid analyzing chip for solving the problem that a PCR method or the like is known as a means for analyzing protein/nucleic acid by extracting the nucleic acid from a specimen but relatively large-sized machinery and peripheral mechanical equipment are required in analysis and the PCR method or the like is not efficient and economical in consideration of that the amount of the specimen is extremely little. SOLUTION: Fine cavities, grooves or the like are formed to one surface of a processable solid plate-like piece such as a glass plate, a silicon plate or the like to form a functional part. This part is used to obtain the analyzing chip capable of continuously performing a series of operation processes from the amplification of the nucleic acid to the examination thereof in a short time.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a protein / nucleic acid analysis chip characterized by efficiently, economically and simply performing the steps required for protein / nucleic acid analysis.

[0002]

2. Description of the Related Art Conventionally, relatively large-sized equipment and peripheral equipment are often used for protein / nucleic acid analysis, and it is inefficient in view of the fact that a very small amount of sample is usually used for protein / nucleic acid analysis. It was inevitable that it was sensible and uneconomical.

For example, P widely used for nucleic acid amplification
In the case of the CR method, in principle, it is possible to analyze from one copy of the gene, but the PCR device requires a large reagent volume in order to use a tube, and as a result, requires a large amount of nucleic acid. And Naturally, the power consumption for operating the device also increases.

In the case of electrophoresis, especially in the case of sequencing, a gel plate of several tens of centimeters is used and a voltage of 3000 V is required. However, considering that 1 μg or less of nucleic acid is used as a sample, such an analysis method is not suitable. It must be said that it is efficient and uneconomical.

In order to make up for the above-mentioned drawbacks, a new nucleic acid test method using a microfabrication technique such as a DNA chip or a nanosequencer has been devised, and a test method for analyzing a very small amount of sample efficiently and economically. Development is in progress.

[0006]

However, each of the minute sample analyzers such as DNA chips, which have been developed up to now, have a single function, and have a function of performing gene analysis comprehensively and efficiently. I don't have it. For this reason, conventional equipment is used for any step in the genetic test to obtain analysis results through a series of steps, and as a result, it is not out of the scope of inefficient and uneconomical test methods. .

[0007] For example, when performing gene analysis by the PCR method, it is roughly divided into four steps of nucleic acid extraction from a sample, nucleic acid amplification by PCR method, electrophoresis of nucleic acid amplification product, and nucleic acid detection by staining. However, since each step uses an independent device and moves the specimen sample, continuous work cannot be performed. In order to perform efficient and economical inspection, it is necessary to miniaturize all of these steps and to make each step continuous.

[0008]

In view of such circumstances, as a result of earnest research, the inventor has conducted a series of these operation steps continuously on a miniaturized analysis chip, resulting in a small amount of reagents and samples. The invention of an epoch-making method for carrying out a sample in a short time with only one small device has been achieved.

A feature of the present invention is that the surface treatment, sterilization treatment, cleaning, etc. of a glass plate, a silicon plate, etc. can be performed, and one surface of a solid plate-like piece capable of transmitting light, such as fine depressions and grooves, is formed. By constructing shapes, functionalizing these shapes to give continuity as parts, and arranging a valve mechanism that opens and closes by heating or cooling in each part, the temperature change of functional parts and valves The non-contact heating and cooling method employs contact cooling to design and manufacture a protein / nucleic acid analysis chip for temperature control.

The shape of depressions, grooves, etc. constructed on the surface of the solid plate-like piece is functionalized to form an analysis chip, and functional parts such as PCR tank, electrophoresis groove, buffer solution tank and valves on the analysis chip are non-functional. A laser beam is used as a heat source for heating by contact,
Infrared rays, far infrared rays, and the like can be used, but the present invention is not limited thereto, and any one can be used as long as it can heat only the target component or valve in a non-contact manner in a short time. In addition, as a means for rapidly cooling the target component or valve by contact, when an electric current is passed through the joint surface of two kinds of metal or semiconductor, heat generation or heat absorption is generated in the joint portion of the electronic device utilizing the Peltier effect. It is suitable for use as a exchanger.

In order to open and close the valve on the analysis chip by changing the temperature, when heated above a certain temperature, the martensite phase transforms to the austenite phase and Ni-Ti or Cu- which has the property of recovering the shape before the deformation. A shape memory alloy such as Ti, or a shape memory polymer having a property of returning to its original shape by losing a certain deformation produced by an external force by heat treatment or the like is suitable.

A further feature of the present invention is that the specific nucleic acid of interest is detected at each specific position by immobilizing one or more specific probes for detecting nucleic acid on the analysis chip, and the property of the nucleic acid is determined from the position pattern. Is to be able to be estimated easily.

[0013]

BEST MODE FOR CARRYING OUT THE INVENTION An example of the principle of the present invention will be described below with reference to the drawings. In this example, PCR, electrophoresis,
The method for carrying out hybridization and fluorescent nucleic acid detection with a single chip is shown as an example, but the present invention is not limited to this.

In FIG. 1, a specimen sample (nucleic acid) is first injected into a PCR tank constructed on an analysis chip together with a PCR reaction solution. This tank is heated in a non-contact manner by a heat ray source installed in the upper part, and is cooled in a non-contact cooling heat exchanger installed in the lower part except during cooling. With respect to the specimen sample in the PCR tank, PCR reaction proceeds by repeating appropriate heating and cooling, and nucleic acid is amplified.

After the completion of the PCR reaction, the heat ray source heats the valve attached to the PCR tank to open the valve, and the amplified nucleic acid product in the PCR tank can be moved to the electrophoresis groove. The valve is made of a shape memory alloy or a shape memory polymer substance that deforms when heated,
It is deformed by heating and passes the nucleic acid product.

After passing through the valve, when the electrodes are energized, the nucleic acid starts moving from the PCR tank to the buffer tank through the electrophoresis groove. The electrophoresis groove can be filled with agar or a polymer compound in advance, but other resistance substance may be used. PC with these resistance materials
Impurities such as proteins present in the R reaction solution are separated during electrophoresis. Further, the electrophoresis groove can be filled with a buffer solution.

When the nucleic acid moves in the electrophoresis groove and undergoes a hybridization reaction with the nucleic acid detection probe fixed in advance in this groove, the nucleic acid is caught by the nucleic acid detection probe to form a double-stranded nucleic acid. Since this occurs when the target nucleic acid sequence exists, it is possible to confirm the presence or absence of the target nucleic acid sequence by the presence or absence of the reaction. In order to cause this reaction, it is desirable to use it in combination with a method for amplifying single-stranded nucleic acid such as asymmetric PCR.

PCR is performed using an intercalator such as Cybergreen that excites fluorescence when a nucleic acid forms a double strand.
When mixed with the reaction solution or the buffer solution, when the fluorescence is applied from the lower part of the analysis chip, the fluorescence of a specific wavelength is emitted.
The presence of nucleic acids is finally confirmed by detecting this excitation fluorescence, but if several types of probes are fixed in the electrophoresis groove, the properties of the gene are determined in a multifaceted pattern from the fluorescence position information. It is possible to

[0019]

[Examples] Using the blood type determination chip whose conceptual diagram is shown in FIG. 2, the PCR reaction solutions shown in Table 1 were put into the PCR tank of FIG. Show. Note that this usage is an example, and the application of the present invention is not limited to this.

[0020]

[Table 1]

The results obtained using the blood typing chip shown in FIG. 2 are shown. FIG. 3 shows an example of determination for AO type Rh (+), FIG. 4 shows an example of determination for BB type Rh (+), and FIG. 5 shows an example of determination for O type Rh (−). The results were the same as the results determined by the blood typing method of.

[0022]

INDUSTRIAL APPLICABILITY The present invention continuously constructs a series of operation steps from extraction and detection of a target nucleic acid from a specimen sample to one surface of a solid plate-shaped piece such as a glass plate or a silicon plate capable of surface processing The present invention relates to a protein / nucleic acid analysis chip consisting of functional components, and by using the chip, analysis operations can be continuously performed on the chip, so that relatively large equipment and peripheral equipment have been conventionally used. In most cases, it is inefficient considering that the amount of sample sample handled for protein / nucleic acid analysis is extremely small, and inefficient when considering that the amount of sample sample that is handled uneconomically is extremely small. It is possible to efficiently and economically carry out a nucleic acid test that has not been subject to a targeted and non-economical influence.

[Brief description of drawings]

FIG. 1 is a conceptual diagram of the entire protein / nucleic acid analysis chip.

FIG. 2 is a conceptual diagram of a blood typing chip.

FIG. 3 is an example of determination in the case of AO type Rh (+) type.

FIG. 4 is an example of determination in the case of BB type Rh (+) type.

FIG. 5 is an example of determination in the case of O type Rh (−) type.

Continuation of front page (51) Int.Cl. ⁷ Identification code FI theme code (reference) G01N 37/00 102 C12N 15/00 F

Claims (9)

[Claims] 1. A process for forming a fine recess, groove, or other shape on the surface of a solid plate-like piece to functionalize the shape into a component, which is used for singular or single analysis necessary for protein / nucleic acid analysis. A protein / nucleic acid analysis chip having a multi-functionality for simultaneously or sequentially performing a plurality of operation steps. 2. The protein / nucleic acid analysis chip according to claim 1, which has a multifunctional property of performing a single or a plurality of operation steps required for protein / nucleic acid analysis by selecting or varying the operation steps. 3. When the single or plural operation steps required for protein / nucleic acid analysis are selected or varied, the valve has a multifunctional function of carrying out movement of a specimen sample between the steps with a valve. Item 1. A protein / nucleic acid analysis chip according to item 1. 4. The opening and closing of the valve according to claim 3,
This is done by using the characteristics of the material that changes shape with temperature.
A protein / nucleic acid analysis chip, which has a function of allowing a sample sample to pass through when the valve is heated and blocking it when cooled. 5. For the purpose of opening and closing the valve according to claim 4, the heating method has a non-contact heating function using a heat source, and the cooling method has a contact cooling function. A protein / nucleic acid analysis chip characterized by the following. 6. A method for heating a functional component for performing a PCR method in a multifunctional analysis chip for performing a single or a plurality of operation steps required for protein / nucleic acid analysis by selecting or varying the operation steps, The protein / nucleic acid analysis chip according to claim 1, which has a non-contact heating function using a heat source and a contact cooling function as a cooling method. 7. A specific probe for nucleic acid detection is used as a functional component of electrophoresis in a multifunctional analysis chip that performs a single or multiple operation steps required for protein / nucleic acid analysis by selecting or varying it. The protein / nucleic acid analysis chip according to claim 1, which has a function of detecting a specific nucleic acid sequence during electrophoresis by immobilizing one or a plurality of nucleic acids. 8. A nucleic acid detection specific one in a groove filled with a hybridization buffer in a multi-functional analysis chip for performing a single or plural operation steps required for protein / nucleic acid analysis by selecting or varying the operation steps. The protein / nucleic acid analysis chip according to claim 1, which has a function of detecting a specific nucleic acid sequence by immobilizing a single or a plurality of dynamic probes and injecting a sample sample. 9. An electrical chip having one or more specific probes for nucleic acid detection fixed in a multifunctional analysis chip for performing a single or a plurality of operation steps required for protein / nucleic acid analysis by selecting or varying the operation steps. 9. The protein according to claim 7, which has a function of determining the nucleic acid information by arranging a single groove or a plurality of grooves for performing electrophoresis or hybridization, patterning the positional information of the detected nucleic acid.・ Nucleic acid analysis chip.