JP2003130874A - Plastic board for microchip - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a plastic board for a microchip, made out of plastic modified and molded to inhibit the intrusion of the excitation light received by the plastic used in the board for preventing the fluorescence generated from the excitation light from reaching a detector. SOLUTION: A material for making the plastic substantially opaque, or opaque at least in the peak wavelength of the detected fluorescence or at least in the peak wavelength of the radiated excitation light, is included as a barrier, to manufacture the plastic board for the microchip not emitting the fluorescence.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a plastic used as a substrate for microchips such as microarrays and DNA chips.

[0002]

2. Description of the Related Art In recent years, rapid progress has been made in the analysis of DNA sequences by world-wide genome projects, and draft sequences of the human genome have already been published. Along with this trend, various genetic analysis techniques have been developed, which has spurred elucidation of life functions. Especially in the medical field, if a database of gene polymorphisms that dynamically respond to various diseases can be constructed, it will be possible to identify target disease-related gene clusters, and further develop new drugs through custom-made treatment and new drug designs. Is believed to be able to proceed. Under such circumstances, microarrays capable of very small-scale extraction, reaction, detection, etc., and the ability to simultaneously measure a large number of genes or a large number of samples, make microchips such as DNA chips the most powerful technique for gene polymorphism analysis. Chips are receiving attention. Further, in the future, a DNA chip is expected to be a tool for making the most effective treatment by individually deciding a diagnosis, a treatment policy, and a drug to be administered in a medical field where a patient is present. Therefore, it is being developed by various research institutions and companies all over the world, and various measurement methods such as microarrays and D
NA chips are being developed.

As a microchip which is expected to be widely used in this field at an early stage, a DNA chip can be mentioned.
As a general measuring method at the present time, an Affymetrix method (US Pat. No. 5,445,934 etc.) in which various kinds of oligonucleotides are arranged side by side by a photolithographic method on a support such as a silicon wafer, or various kinds of glass on a slide glass is used. Stanford type (US Pat. No. 5,807,522) in which nucleic acids are arrayed and immobilized by spotting,
Hybridize nucleic acids that are duplicated from a sample and labeled with a fluorescent label with nucleic acids that are immobilized on a support by a method in which different types of nucleic acids are immobilized on beads made of glass or plastic that can be identified by hue etc. Then, the fluorescence method is used for detection.

Further, the microarray has been highlighted as a tool capable of very small scale extraction, reaction, detection, etc., for its application and high possibility of performance. Especially in the medical field, in order to quantitatively detect a very small amount of a biochemically active substance sample, 0.1 μm to several mm on the array.
Unit spaces and grooves are engraved, micropumps, electrodes, heating, cooling, gates and other devices for manipulating samples in nl units are arranged, and a microarray equipped with a detection system using the fluorescent labeling method Development is in progress. For example, “LabCh” sold by Agilent Technologies, Inc.
"ip" has begun to be marketed, and is excellent in sensitivity and simplicity in detecting a small amount of DNA and RNA. In addition, immunoassay by microarray, chromatography, environmental analysis, cell culture, etc. are considered possible. Furthermore, it will be applied and developed for the synthesis of biochemically active substances and the system that synthesizes and secretes drugs in the body. Conceivable.

However, many problems still exist in the microchip. Although hundreds to tens of thousands of kinds of DNA are arrayed by spotting on a DNA chip and it is very expensive, the process of manufacturing, transporting and inspecting a slide glass of a Stanford type support which is used relatively often If there is a shock, such as a drop, at the point that it is very easily damaged. The microarray is also processed precisely on the glass by using the advanced fine processing technology, and the manufacturing cost becomes very high.

Therefore, in order to be widely and commonly used in medicine, it is easier to handle and cheaper than at present so that various processes performed from collecting a sample from a patient to performing gene analysis can be carried out easily. In order to solve this problem, these days, plastic molded products are used instead of silicon wafers and slide glasses as substrates that can withstand impacts such as drops and can be supplied at low cost. Attempts are being made to begin. Furthermore, by selecting plastic as the material of the substrate, there is an advantage that various shapes that add the function of a microreactor by microfabrication that can perform extraction, mixing and reaction can be mass-produced by a method such as injection molding. is there.

Even when an electrode or a vibrator is used as a DNA carrier in a DNA chip, plastic is sometimes used as a spacer for fixing nucleic acids (Japanese Patent Laid-Open No. 63-501920). However, when plastic is used as a substrate for a microarray or a DNA chip, the substrate itself emits fluorescence depending on the molecular structure of the substance forming the substrate. The fluorescence emitted from the substrate becomes noise and is mixed with the fluorescence signal emitted by the fluorescent label. This fluorescence is a major factor in lowering detection sensitivity in a detection system using a fluorescent labeling substance.

In an attempt to improve these problems, for example, a plastic polymer that does not emit fluorescence as much as possible, a high molecular polymer that does not have n-electrons or π-electrons, for example, Japanese Patent Laid-Open No. 2001-2001.
A polymer having a cyclic olefin structure alone or a saturated polymer obtained by hydrogenating a copolymer of a cyclic olefin and an α-olefin (hereinafter referred to as a saturated cyclic polyolefin), etc. Attempts have been made to select and use, or to use a fluorescent label that can use a wavelength with low fluorescence intensity in the spectrum of fluorescence emitted by the plastic of the substrate. By making full use of the above method, it has become possible to perform a detection method using a fluorescence method, but fluorescence is emitted more strongly than a substrate made of quartz glass, and it has yet to reach a sufficient solution. There wasn't.

[0009]

An object of the present invention is to modify the material so that the plastic used for the substrate is prevented from entering the excitation light and the fluorescence emitted by the excitation light does not reach the detector. The object is to provide a molded plastic substrate for microchips.

[0010]

DISCLOSURE OF THE INVENTION The inventors of the present invention have made extensive studies as to how to solve these problems, and as a result, have made plastic substantially opaque, or at least the peak wavelength of fluorescence to be detected, or at least irradiation. The inventors have found that a plastic substrate that does not emit fluorescence can be manufactured by containing a substance that makes it opaque at the peak wavelength of excitation light, and has completed the present invention.

That is, according to the present invention, (1) a plastic substrate used in a method for detecting a sample treated with a fluorescent label with fluorescence emitted by supplying excitation light is used. A plastic substrate for a microchip, characterized by containing a substance which makes it substantially opaque, (2) a sample which has been subjected to a treatment to which a fluorescent label is added, In the plastic substrate used in the detection method, the plastic contains a substance that makes the plastic substantially opaque at least at the peak wavelength of excitation light to be irradiated or at least the peak wavelength of fluorescence to be detected. Plastic substrate for chips,
(3) The opaque substance is a pigment (1) or (2)
The plastic substrate for a microchip described in (4), the plastic substrate for a microchip according to (3) in which the pigment is a black pigment, (5) the content of the opaque substance is 1 to 6
The plastic substrate for microchip according to any one of (1) to (4), which is 0% by weight, (6) the plastic for microchip according to any one of (1) to (5), wherein the plastic is a saturated cyclic polyolefin resin. The substrate.

[0012]

BEST MODE FOR CARRYING OUT THE INVENTION The "substrate" described in the present invention refers to a member such as a microarray or a DNA chip which is minutely processed or modified on a scale of 0.1 μm to several mm.
The shape is not limited to a flat plate, and may be a sphere, a cylinder, a rectangular parallelepiped, a polyhedron, a flexible sheet, fine particles, or any other special shape. It is possible to use various molding materials for the plastic that is the main raw material of the substrate, but according to the application of the microchip to be manufactured, processing, characteristics of sample and solvent, moldability, heat resistance, chemical resistance , The adsorbability, etc. are taken into consideration. For example, polystyrene, polyethylene, polyvinyl chloride, polypropylene, polycarbonate, polyester, polymethylmethacrylate, polyvinylacetate, vinyl-acetate copolymer, styrene-methylmethacrylate copolymer, acrylonitrile-styrene copolymer, acrylonitrile. −
Butadiene-styrene copolymer, nylon, polymethylpentene, silicone resin, amino resin, polysulfone,
Fluorine resin, saturated cyclic polyolefin resin, etc. are considered.

Excitation light and fluorescence described in the present invention include ultraviolet rays and infrared rays in addition to visible light. The barrier used in the present invention is a substance which is substantially opaque or opaque at least at the peak wavelength of excitation light or fluorescence, and by containing it in the plastic, it is possible to obtain substantially the same surface characteristics as the plastic before the inclusion. If possible, there is no particular limitation. The substance contained as a barrier includes a pigment, preferably a black pigment, and more preferably carbon black from the viewpoints of colorability, heat resistance, bleed resistance and chemical resistance.

In order to detect a fluorescent label added to a very small amount of sample on a microchip, a technique of irradiating strong laser light as excitation light to increase the amount of fluorescence is often used. 1 pigment is added to the plastic
If it is less than 10% by weight, the opacity is insufficient, the laser light is not shielded and penetrates inside the plastic, and the background fluorescence noise generated by the plastic leaks to the outside, and the value is sufficiently large for the signal of the fluorescent label. To be detected. For this reason, at least 1% by weight or more of pigment is required in order to prevent laser light from entering the inside of the surface of the substrate and to prevent the fluorescence generated in the surface molecular layer of the substrate from leaking from the surface molecular layer. By increasing the content of the pigment, it is possible to reduce the surface molecular layer of the substrate that leaks fluorescence and reduce the background fluorescence amount.

When the pigment is blended in an amount of more than 60% by weight, the amount of plastic contained in the substrate decreases,
Moldability, hardness, heat resistance, wettability, sample adsorbability, and other physicochemical properties inherent in plastics may change significantly, which may cause problems in use and is not suitable. If the resin is blended in at least 60% by weight, it is expected that the physicochemical properties of the plastic will not change so much. As a matter of course, the smaller the content of the pigment, the smaller the change in physicochemical properties as compared with the original plastic, which is preferable in designing a microchip.

Considering the above points, the content of the pigment contained in the plastic of the substrate is 1 to 60% by weight, preferably 1.5 to 20% by weight, more preferably 2 to 5% by weight. It is possible to detect fluorescence-labeled signals clearly by reducing the fluorescence generated from plastics without changing the physicochemical properties of plastics practically. In particular, when a saturated cyclic polyolefin resin is used as the main raw material of the substrate, the fluorescence emitted by the plastic itself is less than that of other plastics. Therefore, by performing the formulation described in the present invention, the fluorescence detector It is possible to fabricate a plastic substrate for microchips that hardly detects fluorescence.

There is no particular limitation on the method of incorporating the pigment into the plastic. Depending on the pigment, it is possible to directly compound and knead a low molecular weight olefin polymer. Further, the pigment may be mixed in a stabilizer wax containing a low molecular weight olefin polymer or stearic acid at a high concentration, and then mixed into a plastic as a main raw material so as to have an appropriate concentration and kneaded. .

[0018]

EXAMPLES Next, examples of the present invention will be described. Example 1 A pellet containing a high concentration of a black pigment was prepared by blending 50% by weight of a wax for a stabilizer with 50% by weight of carbon black. A saturated cyclic polyolefin resin colored black was prepared by blending 5% by weight of the above pellets with 95% by weight of a hydrogenated product of a random copolymer of ethylene and dicyclopentadiene. Then, a slide plate having a thickness of 1 mm was injection-molded using this resin.

(Example 2) 5% by weight of the pellet described in Example 1 was mixed with 95% by weight of polystyrene to prepare polystyrene colored black. Then, a slide plate having a thickness of 1 mm was injection-molded using this resin.

Comparative Example 1 A slide plate having a thickness of 1 mm was injection molded using a random copolymer of ethylene and dicyclopentadiene. Comparative Example 2 A slide plate having a thickness of 1 mm was injection-molded using polystyrene. Comparative Example 3 A quartz glass slide glass (S1111, manufactured by Matsunami Glass Co., Ltd.) was used.

The slide plates of Examples 1 and 2 and Comparative Examples 1, 2 and 3 were observed with an epi-illumination fluorescence microscope (excitation light wavelength 532 nm, fluorescence wavelength 560 nm) with no sample or slide placed thereon. Fluorescence was photographed with a CCD camera and the amount of fluorescence was compared and evaluated from the image data. The evaluation results are shown in Table 1. From this, it was confirmed that the background fluorescence can be suppressed by applying the formulation of the present invention.
In particular, it was confirmed that Example 1 had a lower background value than the quartz glass of Comparative Example 3.

[0022]

[Table 1]

[0023]

The prescription of the present invention is used for a microchip by blocking the entry of excitation light received by the substrate and modifying and molding the material so that the fluorescence emitted by the excitation light does not reach the detector. It is possible to manufacture plastic substrates that do not fluoresce.

Front page continuation (51) Int.Cl. ⁷ Identification code FI theme code (reference) G01N 37/00 102 C12N 15/00 FF term (reference) 2G043 AA01 BA16 CA09 DA02 EA01 FA01 FA02 GA03 GB16 KA02 MA01 4B024 AA11 AA20 HA11 4B029 AA08 AA23 CC08 GA03 GB09

Claims (6)

[Claims] 1. A plastic substrate used in a method for detecting a sample treated with a fluorescent label with fluorescence emitted by supplying excitation light, whereby the plastic is substantially opaque. A plastic substrate for a microchip, which contains a substance. 2. A plastic substrate used in a method for detecting a sample treated with a fluorescent label with fluorescence emitted by supplying excitation light, wherein at least the excitation light irradiated by the plastic is A plastic substrate for a microchip, which contains a substance which is substantially opaque at a peak wavelength or at least a peak wavelength of fluorescence to be detected. 3. The opaque substance is a pigment.
Alternatively, the plastic substrate for a microchip according to claim 2. 4. The plastic substrate for a microchip according to claim 3, wherein the pigment is a black pigment. 5. The plastic substrate for a microchip according to claim 1, wherein the content of the opaque substance is 1 to 60% by weight. 6. The plastic substrate for a microchip according to claim 1, wherein the plastic is a saturated cyclic polyolefin resin.