JP2003028867A - Reaction chip and carrier for the same - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a carrier for a reaction chip which is hardly cracked, superior in handleability, and capable of easily controlling the amounts of a reactive substance to be carried and reducing background noise when measuring substances in a specimen by fluorescence analysis. SOLUTION: In the carrier for the reaction chip, the reactive substance such as DNA, RNA, a protein, an antibody, an enzyme, or the like which reacts with the substances is carried in a surface for detecting the substances in the specimen. The carrier for the reaction chip is formed of polyolefine.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a reaction chip carrier used for a reaction chip such as a biochip, and the reaction chip.

[0002]

2. Description of the Related Art Biochips such as DNA chips are
Conventionally, it was produced using a glass plate. However, glass plates are fragile and difficult to handle. Further, there is a problem that it is difficult to control the amount of DNA or protein immobilized on the surface of the glass plate.

Therefore, Japanese Unexamined Patent Publication No. 2000-295990 discloses a DNA chip in which DNA is bound to the glass surface by spotting an aqueous liquid containing a DNA fragment and a hydrophilic polymer on the surface of the glass plate. There is.

Further, Japanese Unexamined Patent Publication No. 10-248599 discloses a reaction chip capable of supporting a reactive substance such as DNA at a high density on a carrier at a laboratory level. Here, a stretchable substrate made of rubber or the like is stretched in a one-dimensional or two-dimensional direction, and in the stretched state of the stretchable substrate, a reactive substance is carried on minute sections of the stretchable substrate surface.
After that, the stretchable substrate is shrunk to the size before stretching, whereby a reaction chip carrying a large number of reactive substances such as DNA fragments can be obtained.

[0005]

As described above, the reaction chip using the conventional glass plate is easily broken.
It was difficult to handle. Also, JP-A-10-24859
When a stretchable base material such as rubber is used as a carrier like the reaction chip described in Japanese Patent Publication No. 9, since a large amount of fluorescent substance is contained, background noise may be generated during detection using a fluorescence analysis method. There was the problem of becoming expensive. That is, it has been difficult to analyze a substance in a sample with high accuracy using a reaction chip.

The object of the present invention is to prevent cracking, to be easy to handle, to easily control the amount of the reactive substance to be carried, and to reduce the background noise when measuring by the fluorescence analysis method. (EN) It is possible to provide a carrier for a reaction chip and a reaction chip capable of detecting a substance in a sample with high sensitivity.

[0007]

The carrier for a reaction chip according to the present invention is a carrier for a reaction chip in which a reactive substance that reacts with a substance for detecting a substance in a sample is carried on the surface. And polyolefin.

In a particular aspect of the invention, the polyolefin is polyethylene or polypropylene. Preferably, the reaction chip carrier is configured to be black or transparent.

[0009] In another particular aspect of the present invention, there is provided a carrier used in a reaction chip in which the substance in the specimen is detected by fluorescence analysis, that is, the substance is detected by fluorescence analysis.

A reaction chip according to the present invention comprises a reaction chip carrier constructed according to the present invention and a reactive substance carried on the surface of the reaction chip carrier. Hereinafter, the details of the present invention will be described.

The carrier for a reaction chip according to the present invention is made of polyolefin. Examples of the polyolefin include polyethylene, polypropylene, polybutene, polymethylpentene, and the like. In the case of fluorescence measurement, polyethylene or polypropylene is preferably used because a material that emits fluorescence or reflects light is not preferable. The grade such as polyethylene or polypropylene is not particularly limited, and for example, both high density polyethylene and low density polyethylene can be used.

In the present invention, "reactivity" in "reactive substance" means not only a case where a chemical structure is changed by an ionic bond or a covalent bond due to a chemical reaction, but also a hydrogen bond,
It includes a wide range of properties capable of forming a state of being bound to another substance by various modes such as coordination bond, van der Waals force, chemisorption, and physical adsorption. Examples of such reactive substances include, but are not limited to, DNA fragments, enzymes, antigens, solids, epitopes, proteins and the like.

Further, the substances in the sample detected by using the reaction chip according to the present invention shall widely include various substances which react with the above-mentioned reactive substances, such as DNA, enzyme,
Organisms for enzymes, antigens, solids, epitopes, proteins and the like can be mentioned.

The shape of the carrier for a reaction chip according to the present invention is not particularly limited and may be plate-shaped or any shape other than a plate, but it is generally used for constituting a reaction chip. A plate-like shape is preferable. When the plate is configured, the structure is not particularly limited, and it may be a single plate type plate made of a single polyolefin or a laminated type plate in which a plurality of polyolefin layers are laminated.

The surface of the reaction chip carrier according to the present invention is preferably subjected to corona discharge treatment. Corona discharge treatment is a type of plasma discharge, in which high frequency and high voltage are applied to the polyolefin surface with few polar groups to generate corona discharge in the atmosphere, and the functional groups, electrons and polyolefins generated by this are given to the surface. Is the way to do it.

In the corona discharge treatment, however, when the applied power becomes too large, the surface of the carrier for the reaction chip becomes physically rough, and holes are formed on the surface of the carrier or the roughened surface is formed. Dust may accumulate, which may interfere with observation.

The carrier for the reaction chip according to the present invention is preferably subjected to hydrophilic treatment. The method of this hydrophilic treatment is not particularly limited, and it can be carried out by applying a coating such as corona discharge or a surfactant to a carrier made of polyolefin. Regarding the degree of hydrophilic treatment, it is preferable that the wetting tension is in the range of 35 dyne / cm to 95 dyne / cm. Wetting tension is 35 dyne / c
When it is less than m, the hydrophilicity is lower than that of glass, static electricity is apt to be applied, and dust and dirt are easily attached. Further, in the case of a liquid sample, the spread of the sample on the reaction chip carrier may be poor, and observation may be difficult. On the other hand, if it exceeds 95 dyne / cm, ionic dust in the air is likely to collect due to the influence of water vapor or the like, and the contamination of the carrier progresses, which may hinder the observation.

The reaction chip carrier has a thickness of 40.
It may be a film having a thickness of 0 μm or less. When it is made into a film, it is easy to improve the coating efficiency when coating the surface with DNA or protein, and especially when the reactive substance is sprayed and fixed on the carrier, the fixing rate can be increased. However, it is preferable.

In the reaction chip according to the present invention, the reactive substance is carried on the surface of the carrier for the reaction chip, but the carrying method is not particularly limited. That is, the term broadly includes a method of chemically bonding a reactive substance to the surface of a carrier, a method of adsorbing the reactive substance on the surface of the carrier, a state of placing the reactive substance on the surface of the carrier, and the like.

The method for detecting a substance in a sample using the reaction chip according to the present invention is not particularly limited, but a fluorescence analysis method is preferably used. That is, the substance in the fluorescently labeled sample is flown onto the reaction chip, the substance in the sample reacts with the reactive substance carried on the reaction chip, and is immobilized on the reactive substance. Then, the fluorescently labeled substance in the sample immobilized on the reactive substance on the reaction chip carrier is detected by a fluorescence analysis method. In this case, since the carrier for the reaction chip is made of polyolefin and the content of the fluorescent substance in the polyolefin is very small, the substance in the sample can be detected with high accuracy by the fluorescence analysis method.

[0021]

BEST MODE FOR CARRYING OUT THE INVENTION The present invention will be described in more detail by giving specific examples of the present invention.

(Production of Plate) As shown in FIG. 1, a rectangular black plate 1 having a planar shape of 2.5 cm × 8 cm.
Were manufactured using various materials shown in Table 1 below.

Fluorescence intensity of the plate made of each of these materials was measured using a ScanArray4000 manufactured by BM Kikai Co., Ltd. at a laser output of 100%, a detection gain of 100%, an excitation light wavelength of 550 nm, and an emission wavelength of 570 nm. The results are also shown in Table 1 below.

[0024]

[Table 1]

Based on the results shown in Table 1 above, each plate made of low density polyethylene (LDPE), high density polyethylene (HDPE) and polypropylene having a low fluorescence signal intensity was used, and DN as a reaction chip was prepared by the following method.
An A chip was produced.

(Preparation of DNA Chip) Thirty plates were placed in a rack and submerged in a 0.1N sodium hydroxide aqueous solution. Shake for 30 minutes at room temperature and rinse the plate with distilled water.

70 mL of poly-L-lysine (manufactured by Sigma) was added to 280 mL of distilled water to prepare a diluted solution of poly-L-lysine. The above-mentioned 30 plates were immersed in the above poly L-lysine diluted solution together with the rack and left for 1 hour, and then the whole rack was centrifuged at 500 rpm for 1 minute. In this way, the diluted poly-L-lysine solution was removed, and then dried at 40 ° C. for 50 minutes.

(DNA spotting) A 3 × SSC (standard salt-citrate buffer) aqueous solution of a PCR product which is a yeast gene fragment consisting of about 2000 base pairs into which an amino group has been introduced (the concentration of the PCR product is 0.5 mg. / ML) into a 96-well microtiter plate. After adding 3m sodium acetate in 1/10 volume of the PCR product 3 × SSC aqueous solution and isopropanol in the same amount as the PCR product 3 × SSC aqueous solution, the mixture was left standing at −20 ° C. for 3 hours,
Centrifuge at 3500 rpm for 45 minutes to precipitate the DNA,
Then the supernatant was removed.

The above precipitate was washed with 70% ethanol and centrifuged at 3500 rpm for 45 minutes, after which the supernatant was removed. The precipitate was then dried at room temperature. To the above precipitate, add 12 μL of 3 × SSC solution and leave it for 3 hours.
NA was completely dissolved. DNA thus obtained
Transfer the solution to a U-bottom 96-well microtiter plate,
It was set in an array machine (GMS417 Arrayer, manufactured by Takara Shuzo) and spotted on the plate prepared as described above.

(Post-Spotting Treatment) A plate on which spotting was completed was placed on a tray containing distilled water at 60 ° C. with the surface on which DNA was attached facing down and held for 1 minute. After that, the plate was placed on a hot plate at 95 ° C. with the surface having the DNA attached thereon facing upward. After that, 60 on the surface with DNA attached
The DNA was crosslinked by irradiating it with UV light at an intensity of mJ.

(Blocking) 5 g of succinic anhydride (Aldrich), 315 mL of N-methyl-pyrrolidone (Aldrich), and 0.2 M sodium borate buffer (pH 8.0) were mixed, and the post-treatment liquid was used. It was made. The post-spotting post-treatment plate was immersed in this post-treatment solution, permeated for 20 seconds, and then deposited for 15 minutes.

Next, the plate was taken out and immersed in distilled water at 95 ° C. for 2 minutes, then taken out from the distilled water and immersed in 95% ethanol for 1 minute. After that, 5
The plate was centrifuged at 00 rpm for 1 minute, and the plate was stored dry at room temperature.

(Preparation of labeled nucleic acid (adjustment of substance to be detected)) Cy5-labeled dCTP was incorporated into a reverse transcription reaction of mRNA extracted from yeast to prepare a fluorescent labeled cDNA.

(Hybridization and measurement) The above-mentioned labeled cDNA (1 mM) was used for hybridization solution (4 × SSC and 10% SDS mixed solution, mixing ratio:
What was dispersed in a volume ratio of 1:10) 20 μL was overlaid on the DNA chip obtained as described above, and incubated in a moisture chamber at 60 ° C. for 20 hours. Next, this was mixed with 0.1 wt% SDS (aqueous solution) and 2
It was immersed in a mixed solution with xSSC (mixing ratio is 1: 1 by volume). Next, after sequentially washing with a mixed solution of 0.1% by weight SDS and 2 × SSC, a mixed solution of 0.1% by weight SDS and 0.2 × SSC, and a 0.2 × SSC solution, 600r
It was centrifuged at pm for 20 seconds and dried at room temperature.

The fluorescence intensity of the surface of the plate thus dried was measured by ScanArray 5000 (manufactured by BM Equipment Co., Ltd.). The results are shown in Table 2 below.

[0036]

[Table 2]

[0037]

The carrier for a reaction chip according to the present invention is made of polyolefin so that it is hard to crack and easy to handle. Further, since polyolefin has a low content of a fluorescent substance, for example, when a substance in a sample is detected by using fluorescence analysis, background noise is reduced and the substance can be detected with high accuracy.

When polyethylene or polypropylene is used as the above-mentioned polyolefin, the content of the fluorescent substance is much smaller, so that the substance in the sample can be measured with higher accuracy.

When the carrier for the reaction chip is black or transparent, the background noise in fluorescence analysis can be further reduced. When the substance in the sample is detected by fluorescence analysis, the substance in the sample can be detected with high sensitivity because the content of the fluorescent substance in the carrier is small.

Since the reaction chip according to the present invention has a structure in which a reactive substance is carried on the surface of the reaction chip carrier constructed according to the present invention, background noise is not detected when detecting a substance in a sample by fluorescence analysis. It can be lowered, the substance in the sample can be measured with high sensitivity, and it is difficult to break, so that it is easy to handle.

[Brief description of drawings]

FIG. 1 is a plan view showing a planar shape of a plate facilitated in an embodiment.

[Explanation of symbols]

1 ... Plate (carrier for reaction chip)

Claims (5)

[Claims] 1. A carrier for a reaction chip, which is a carrier for a reaction chip, in which a reactive substance that reacts with the substance for detecting a substance in a sample is carried on a surface, and is made of polyolefin. 2. The carrier for a reaction chip according to claim 1, wherein the polyolefin is polyethylene or polypropylene. 3. The method according to claim 1, which is black or transparent.
The carrier for a reaction chip according to item 1. 4. A carrier for a reaction chip in which the substance in the sample is detected by fluorescence analysis. 5. A reaction chip comprising the carrier for a reaction chip according to claim 1 and a reactive substance carried on the surface of the carrier for a reaction chip.