JP2000236876A - Polymerase chain reaction device having integrated microwell - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a polymerase chain reaction device having integrated microwells capable of carrying out many DNA analyses at a micro sample by forming an assemblage composed of plural microwells on a semiconductor base plate and composing it so as to carry out polymerase chain reaction in each well. SOLUTION: By initially forming an SiO2 film 6 by means of wet heat oxidation at 1000 deg.C for 8 hours on the surface 5 of silicon wafer of 500 μm thick out off in the size of 30 nm×30 nm, applying a negative type photoresist on the one side by means of spin coating, exposing it through a photomask, removing the excessive resist after being developed, immersing an oxidized film revealed on the surface in an acidic solution to remove it, finally carrying out anisotropic etching and further similar lithograph to form microwell assemblage 1 composed of integrated plural microwells 3 on the semiconductor base plate 2 and composing it so as to respectively carry out polymerase chain reactions in respective microwells, the objective device, the microwell inside 4 of which is hydrophilic and the surface 5 of the base plate is hydrophobic, is provided.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION The present invention relates to a method for preparing a microwell integrated body on a semiconductor substrate and performing a polymerase chain reaction (PCR) in the well to obtain a large number of DNA samples at once with a small amount of sample. The present invention relates to a device capable of performing amplification and analysis.

[0002]

2. Description of the Related Art The PCR method is one of the most important techniques in molecular biology. The PCR method is used for medical microbiology,
It is used for analysis in a wide range of fields such as clinical diagnosis of genetic diseases, screening of genes encoding antibodies, and forensic medicine. In a clinical setting, it is desirable to be able to analyze a larger number of samples more quickly in order to diagnose a wide variety of genetic diseases. In the study of genetic diseases, it is necessary to analyze the genetic information of many people in order to create a gene phylogenetic diagram, but the information contained in the human genome is enormous and an efficient analysis method is needed. is there.

[0003]

However, the conventional PCR
Requires a sample volume of 10 to 100 μl, and a device capable of performing PCR with a smaller amount of sample accurately is required. In addition, since it is difficult to amplify and analyze a large number of DNA samples at the same time, a high-density and small-sized apparatus capable of processing a large number of samples at once is required.

[0004]

Accordingly, a high-density microwell integrated body of silicon chips was manufactured by using a semiconductor microfabrication technique. The microwell integrated in such a manner has properties suitable for performing PCR in the well, and it has become possible to simultaneously measure a large number of samples. P
Confirmation of CR was performed by using a fluorescent indicator. Furthermore, by covering the microwells with a membrane that allows water vapor to pass through but does not allow liquid to pass through, the products after PCR do not mix with each other, making it possible to use samples after the reaction.

[0005]

[Example] (Method of manufacturing microwell integrated body) 30 m
500μm thick silicon cut to mx30mm
Wet thermal acid at 1000 ° C for 8 hours on the surface of wafer
SiO_TwoA film was formed. This one side has a negative
Spin-coated photoresist (4000 rpm, 20
Second) and exposed through a photomask (8
Seconds). After development, excess resist was removed and appeared on the surface
Oxide film is 1: 6 HF / NH_FourImmerse in F solution for 5 minutes
Removed. After removing the remaining resist, TMA
H (tetramethoxyammmoniumhydroxyrate) solution
At 80 ° C. for 1 hour.
Was. Again, wet thermal oxidation at 1000 ° C for 4 hours
After that, using a positive photoresist,
Perform photolithography, 1: 6 HF / NH _FourF solution
The oxide film other than the inner wall of the chamber
Removed. Of the microwell assembly thus produced
A cross-sectional view is shown in FIG. On microwell assembly (1)
Has a pyramid with a length of 50 μm, a width of 50 μm, and a height of 27 μm.
Form microwells (3) at 50μm intervals
And a silicon chip within 2 cm square
40,000 wells were arranged. The volume of each well is 23 pl
And a well with a capacity of 1/200000 of a normal PCR is created.
Was made. On the base (2) of the microwell assembly (1)
The inner wall (4) of the well (3) prepared in (1) is an oxide film (6).
Hydrophilic because it is covered and the substrate surface (5)
Is so hydrophobic that the well (3) is in it.
And has properties suitable for performing PCR.

(Conditions for PCR) Green Fluor
The GFP gene was amplified using the plasmid into which the escape protein (GFP) gene was introduced as a template. After preparing a solution having the composition shown in Table 1 and injecting the solution into the microwell, all the wells were covered with a glass plate and sealed, and PCR was performed using a thermal cycler. The thermal cycle condition is 95
After preheating at 2 ° C. for 2 minutes, 40 cycles of 94 ° C. and 65 ° C. were performed for 1 minute each. The following two types were used as primers and probes. Forward primer: GTGCTGCCATACACCAT
GAGTG reverse primer: ATCCCCCATGTTGTGGC
AAAA probe: Fam-CGGCCACTACTTCTCT
GACAACGATCG-Tamra

[0007]

Table 1 Composition of solution in PCR Reagent Concentration GFP 1.35 ng / ml Each primer 0.5 μM TaqMan probe 0.63 μM bovine serum albumin 0.2% w / v dNTP 200 μM Tris-HCl (pH 8.3) 10 mM KCl 50 mM MgCl ₂ 5.5 mM AmpliTaq Gold 0.025 U / μl Sterile water −

(Confirmation of PCR) The amplification of the target DNA was confirmed by TaqMan chemistry, which is a method of indicating by fluorescence. The fluorescence intensity was measured using a microscope
Observed with a CD camera. As a result, compared with the fluorescence microscope image (FIG. 2) of the microwell before performing PCR,
In the fluorescence microscope image (FIG. 3) after performing PCR, the fluorescence intensity increased, and amplification of the GFP gene was confirmed. FIG. 4 shows a three-dimensional graph of the fluorescence intensity before and after performing PCR.
Shown in

[0009] In order to prevent the sample from being mixed due to the capillary effect when the cover glass is removed, a HIPORA film is used. FIG. 5 shows the structure of the entire microwell assembly including the HIPORA film and the cover glass. Since the HIPORA membrane (8) has many micropores, liquid water cannot pass through, but water vapor passes through the membrane. When only the cover glass (7) is removed after the PCR, the microwell integrated body (1) is covered only with the HIPORA film (8), and it becomes possible to heat and evaporate the water of the PCR sample. Because of drying, the sample is not mixed due to the capillary effect.
After the CR, the sample can be used by removing the HIPORA film (8).

[0010]

By using the microwell integrated on the silicon chip according to the present invention, it has become possible to carry out a PCR reaction of a large number of DNA samples simultaneously with a small amount of sample.

[Brief description of the drawings]

FIG. 1 is a cross-sectional view of a microwell integrated body.

FIG. 2 is a fluorescence microscopic image of a microwell before PCR.

FIG. 3 is a fluorescence microscopic image of a microwell after PCR.

FIG. 4 is a three-dimensional graph of fluorescence intensity before and after PCR.

FIG. 5 shows the overall structure of a microwell assembly including a HIPORA film and a cover glass.

[Explanation of symbols]

1 microwell assembly, 2 microwell substrate,
6. 3 microwells, 4 microwell inner walls, 5 substrate surface, 6 oxide film, 7. Cover glass, 8HIPOR
A film

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[Procedure amendment]

[Submission date] December 20, 1999 (1999.12.
20)

[Procedure amendment 1]

[Document name to be amended] Statement

[Correction target item name] Claims

[Correction method] Change

[Correction contents]

[Claims]

 ──────────────────────────────────────────────────続 き Continuing on the front page (72) Inventor Yuji Murakami 1--50 Asahidai, Tatsunokuchi-cho, Nomi-gun, Ishikawa Prefecture Staff room D-33 (72) Inventor Toshifumi Sakaguchi 1-50 Asahidai, Tatsunokuchi-cho, Nomi-gun, Ishikawa Staff D Room No. 13 (72) Inventor Morita Yoshitaka Ishikawa Prefecture Nomi-gun Tatsuguchi-cho Miyatake Riki-67 (72) Inventor Nagai Hidenori Nomi-gun Ishikawa Prefecture Nomi-gun Tatsuguchi-cho Asahidai 1-8 Student Dormitory 3-514 Room (72) Inventor Ide Kotaro Kami 1-50 Asahidai, Tatsunokuchi-cho, Nomi-gun, Ishikawa Prefecture Student dormitory Room 3-405 F-term (reference) 4B024 AA11 AA20 BA80 CA01 HA19 4B029 AA08 BB20 GA03 GB04 GB09 4B063 QA01 QA13 QA17 QA18 QQ42 QR08 QR62 QS25 QX02

Claims (7)

[Claims] An apparatus for performing a polymerase chain reaction method, wherein a microwell integrated body including a plurality of integrated microwells is formed on a semiconductor substrate, and the integrated body is constituted. A polymerase chain reaction apparatus characterized in that a polymerase chain reaction is performed in each microwell. 2. The polymerase chain reaction device according to claim 1, wherein an inner wall surface of each of the microwells is hydrophilic, and a surface other than the inner wall surface of the microwell is hydrophobic. 3. The polymerase chain reaction device according to claim 2, wherein an oxide film is provided on an inner wall surface of each of the microwells, and the oxide film on a surface of the semiconductor substrate is removed. 4. A film for covering the microwell integrated body side of the semiconductor substrate, wherein the film is permeable to water vapor but impermeable to liquid, and the film is used for each polymerase chain reaction in each microwell. 2. The polymerase chain reaction device according to claim 1, wherein mixing of the products is prevented. 5. A method for performing a polymerase chain reaction, comprising: using a semiconductor substrate on which a microwell integrated body including a plurality of integrated microwells is formed; Wherein the polymerase chain reaction is performed. 6. The semiconductor substrate is covered with the microwell assembly side by a film that allows water vapor to pass therethrough but does not allow liquid to pass therethrough, and the product of each polymerase chain reaction in each microwell is dried to form the film. The polymerase chain reaction method according to claim 5, wherein 7. The polymerase chain reaction method according to claim 5, wherein the product in each of the microwells is dried, and then a fluorescent indicator is dropped to detect fluorescence from each of the microwells. .