JP2006349559A - Reaction container and substance detecting method using it - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a reaction container for detection having well-shaped reaction parts, capable of detecting a substance from its base side with good sensitivity without exerting an adverse effect on a reaction system and causing no fluctuations in detection in a plurality of the well-shaped reaction parts, and a substance detecting method using the reaction container. <P>SOLUTION: In the reaction container constituted by providing a well-shaped reaction detecting part to a substrate, the average light transmittance at a wavelength of 400-650 nm of the substrate between the base of the well-shaped reaction detecting part and the back of the substrate is 80% or above and the haze of the substrate is 10 or below. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to a reaction vessel used for antigen detection and DNA detection, for example, by an antigen-antibody reaction, and a substance detection method using the same.

  In recent years, μ-Total Analysis System technology and Lab-on-Chip technology for performing chemical reactions, DNA reactions, protein reactions, etc. on the chip have been researched and realized, and so far large-scale experimental devices and a large amount of reagents are required. It is now possible to perform reaction experiments with a small amount of reagent on a chip of several millimeters or less.

  As such a chip, for example, as a DNA chip, a probe DNA is placed on a slide glass, a sample is allowed to act, and DNA is detected (see Patent Document 1), or a minute hole called a well in glass or the like It has been known that a hollow forms a formation and performs a detection reaction in a well.

  As the well type substrate, for example, a detection substrate having a large number of wells provided on the substrate surface is disclosed (see Patent Document 2).

  Various detection methods of these detection chips are known, but the general method is to use fluorescence detection, and a fluorescence detection unit is provided on the upper surface of the chip where a well-like reaction detection unit exists to detect the detection. Is the method.

  However, the detection from the upper surface, when there are multiple well-like reaction detection units, if the amount of the reaction solution in each well is not constant, the distance from the liquid level to the detection unit will vary and accurate data cannot be obtained Sometimes. When reaction and detection are performed in a well-like reaction detection unit, mineral oil having a lighter specific gravity than the reaction solution may be added to the reaction solution in order to prevent evaporation. In such a case, there is a possibility of being affected by refraction at the interface between the lower layer, which is the reaction solution, and the upper layer made of oil. In particular, if there are variations in the amount of reaction solution and oil, the conditions in each well will change, so accurate data cannot be obtained. Similarly, even if the upper layer liquid is colored or suspended, accurate detection cannot be performed.

  On the other hand, when detecting from the lower layer, the distance from the bottom surface of the liquid to the detection part can be easily set to the same condition in a plurality of wells, but is affected by optical characteristics such as transparency of the substrate. For example, when using a light-absorbing base material such as a light-scattering base material such as a slide glass or a colored base material, the distance from the bottom surface of the reaction solution to the detection unit is the same for multiple wells. However, since the substrate is sandwiched, the fluorescence is scattered or absorbed, and the detection sensitivity is lowered. In addition, when forming a well, irregularities are generated on the surface of the bottom surface depending on the molding method, and light scattering due to the irregularity can also be considered. In addition, the base material has other requirements such as chemical resistance, heat resistance, and a material that does not adversely affect the reaction system, and the selection of the base material and the method of molding the well are limited.

Japanese National Patent Publication No. 11-512293 WO2003 / 031972

  The present invention has been made in consideration of such circumstances, and in a reaction vessel having a well-like reaction part, detection that can be detected from the bottom side of the well-like reaction vessel with high sensitivity without adversely affecting the reaction system. An object is to provide a reaction vessel for use. It is another object of the present invention to provide a detection reaction container having no variation in detection in a plurality of well-like reaction units. Furthermore, it aims at providing the detection method using this reaction container.

  According to the first aspect of the present invention, in a reaction container having a well-like reaction detection unit on a substrate, the average light transmittance at a wavelength of 400 to 650 nm between the bottom surface of the well-like reaction detection unit and the back surface of the substrate is 80%. It is the above and it is a reaction container characterized by haze being in the range of 10 or less.

  The invention according to claim 2 is the reaction container according to claim 1, wherein the distance between the bottom surface of the well-like reaction detector and the back surface of the substrate is 0.3 or more and less than 1 mm.

  A third aspect of the present invention is the reaction container according to the first or second aspect, wherein the bottom surface of the well-like reaction detector is flat.

  A fourth aspect of the present invention is the reaction container according to any one of the first to third aspects, wherein a plurality of the well-like reaction detection units are provided.

  The invention according to claim 5 is the reaction container according to any one of claims 1 to 4, wherein the base material is a cycloolefin resin or a methylpentene resin.

  A sixth aspect of the present invention is the reaction container according to any one of the first to fifth aspects, further comprising a well-like reagent container.

  The invention according to claim 7 is the reaction container according to any one of claims 1 to 6, further comprising a PCR reaction unit.

  In the invention according to claim 8, the substrate has a well-like reaction detecting portion, and the average light transmittance at a wavelength of 400 to 650 nm of the substrate between the bottom surface of the well-like reaction portion and the back surface of the substrate is 80% or more. And a step of injecting a recognition substance into a well-like reaction detector having a haze of 10 or less, and a step of injecting a detection substance into the well-like reaction detector, wherein either the recognition substance or the detection substance is A substance detection method comprising a step of detecting fluorescence from the bottom side of the well-like reaction detection unit, which is fluorescently labeled and detects the presence or absence of a reaction between the recognition substance and the detection substance.

  The invention according to claim 9 is the method for detecting a substance according to claim 7, wherein the detection substance is a nucleic acid.

  According to the present invention, in a detection reaction vessel having a well-like reaction part, the presence or absence of a reaction can be detected with high sensitivity from the well-like bottom side without adversely affecting the reaction system. In addition, the presence or absence of a reaction can be detected without variation in detection in a plurality of well-like reaction parts.

Hereinafter, embodiments of the present invention will be described with reference to the drawings.
FIG. 1 is a diagram showing an embodiment of the present invention. In FIG. 1, a plurality of well-like reaction detectors for reacting a sample and a reagent are formed on a substantially rectangular plate-like substrate.

The substrate used in the present invention may be any substrate that has high transparency and does not adversely affect the reaction system.
As such, for example, PC (polycarbonate), PP (polypropylene), cycloolefin polymer, fluoropolymer, silicon resin, and the like can be used.
Of these, cycloolefin resins (Zeonor (manufactured by ZEON Corporation)) and methylpentene resins (TPX (manufactured by Mitsui Chemicals)) are used in terms of transparency, heat resistance, chemical resistance and influence on the reaction system. It is preferable.

It is preferable to form a substrate using such a synthetic resin because it is excellent in heat resistance, chemical resistance, moldability, and the like. Further, two or more kinds of resins may be joined and used. In this case, by creating a substrate by taking advantage of the characteristics of each resin, it becomes possible to make various substrates according to the characteristics of the reagent, the sample, and the like, which can be used for each application. For example, it is possible to divide the material between the upper half and the lower half of the substrate. In addition, the material can be divided into parts such as a reagent storage section and a PCR reaction section described later.
In addition, you may use glass as a raw material of a board | substrate.

The substrate includes a well-like reaction detection unit.
The well-like reaction detector can be formed by cutting or molding if the base material is plastic or synthetic resin. If it is glass, it can be formed by cutting.
Further, a plurality of well-like reaction detection units can be provided.

  In the present invention, the average light transmittance at a wavelength of 400 to 650 nm of the substrate between the bottom surface of the well-like reaction part and the back surface of the substrate is 80% or more for highly sensitive light emission detection from the lower direction of the substrate, and It is necessary that the haze is within the range of 10 or less.

  The fluorescence wavelength in fluorescence detection is mainly between 400 and 650 nm, and the average light transmittance in this range is 80% or more, preferably 85% or more. If it is less than 80%, the light emission intensity at the time of detection is lowered, the influence of the background is increased, and detection with high accuracy becomes difficult. For example, each reaction in the life science field, such as DNA-related technology, uses a small amount of sample and uses a very small amount of fluorescent substance at the μl level. Therefore, it cannot be expected to be so strong compared to other fields. If the original light emission intensity is strong, detection is possible even if the light transmittance of the substrate is low, but in the case of a fluorescent substance used for analysis in the life science field, it is preferably within the above-mentioned range.

  The light transmittance is preferably within the above range, but the light transmittance within a range of ± 30 nm of the wavelength of excitation light and the fluorescence wavelength for detection may be 80% or more, preferably 85% or more. .

  Examples of fluorescent labeling substances used in the life science field include FAM (excitation wavelength 470 nm, fluorescence wavelength 520 nm) and RED (excitation wavelength 570 nm, fluorescence wavelength 620 nm).

  If the haze is greater than 10, light emission reaching the detection unit due to scattering is reduced, and high-sensitivity detection becomes difficult. In addition, haze here says the average value measured by three different points.

  The well shape is preferably flat at the bottom. If the bottom tip has a spherical shape or a conical shape with a sharp point, light is refracted or scattered and cannot be detected with high sensitivity.

  Further, the center line average roughness (Ra) of the surface of the well bottom is preferably low. The above-mentioned haze is related to the properties and film thickness of the substrate itself. However, if the substrate has the same properties and film thickness, the lower the Ra, the lower the haze value. Specifically, the center line average roughness (Ra) is less than 5 μm, preferably less than 1 μm. The center line average roughness (Ra) can be measured using a known laser microscope.

Further, in consideration of the injectability of the reaction solution such as mixing of bubbles, a truncated cone shape in which the wall surface is inclined from the well opening to the bottom is preferable. With such a shape, the liquid can be injected without mixing bubbles.
In addition, the size of the well is not particularly limited, but in the life science field, reaction and detection in a very small amount are often performed, and the diameter and depth of the opening is 5 mm or less, particularly 0.01 mm to 5 mm. It may be within the range.
Moreover, it is preferable that the inner surface of a well-like reaction detection part is smooth. This is because optical properties such as haze and light transmittance are improved at the bottom, and liquid filling is facilitated at the side.

  Moreover, it is preferable that the distance (refer FIG.1 (b)) from the bottom part of a well to the back surface of a base material is 0.3 mm or more and less than 1 mm. If it is less than 0.3 mm, deformation may occur due to heat during reaction. When it is 1 mm or more, the transparency is lowered and the detection sensitivity is lowered. If it is within the above range, the number of miss shots due to molding is reduced, which is preferable.

Moreover, in order to make it easy to fill the well-like reaction detector with the test solution, a hydrophilic treatment may be performed. Specifically, the contact angle with pure water is less than 60 degrees, preferably less than 30 degrees. As such a hydrophilic treatment, for example, atmospheric pressure plasma treatment can be performed.
The contact angle is measured using a known contact angle meter, and the reaction solution is filled using a dispenser, a syringe, a syringe, or the like. Further, the hydrophilization treatment is not limited to the atmospheric pressure plasma treatment, and may be performed by corona treatment or coating treatment.

  In addition, a reagent container may be provided on the same substrate (see FIGS. 2, 3, and 4). The reagent container can be formed in a well shape, and there is no particular limitation on the size. A plurality of reagent storage units can be provided according to the type of reagent used. For example, the reagent container contains a solution containing a detection substance and a plurality of recognition substances, and when performing a multi-step reaction, a solution containing one kind of recognition substance, or other buffers, diluents, etc. it can.

In addition, when used for DNA detection reaction, a PCR reaction unit may be provided on the same chip (see FIG. 3).
By providing a PCR reaction section, sample preparation and DNA detection can be performed on the same chip.
As the PCR reaction part, a well-like reaction part may be provided, or a flow path may be provided to carry out the reaction in the flow path.
Moreover, you may provide another reaction part.

  Moreover, you may provide the flow path which connects well-like reaction detection parts (refer FIG. 4). Further, a flow path connecting the well-like reaction detection unit, the reagent storage hole, the PCR reaction unit, and other reaction units may be provided. By forming these flow paths, it is possible to perform a continuous reaction. As a result, the inspection time can be shortened, various analyzes can be performed with a small amount of sample and reagent, and the cost can be reduced.

The reaction container of the present invention can be used for various biochemical reactions, for example, for detection of antigen-antibody reaction and DNA reaction.
In the present invention, the detection substance is a target substance to be detected, and the recognition substance is a substance used for specifying the detection substance.
In the case of antigen detection by antigen-antibody reaction, for example, a sample containing an antigen as a recognition substance is placed in each well-like reaction detection unit in advance, and then a reagent containing an antibody is added as a detection substance. The presence or absence of reaction can be detected by attaching a labeling substance to any of the above. As the labeling substance, a luminescent substance such as fluorescence is generally used. In this case, a reagent storage unit may be provided on the substrate to store the detection substance.

  In the case of detecting DNA, for example, a nucleic acid probe is prepared in advance in the well-like reaction detection unit as a recognition substance. Next, sample DNA can be supplied to the well-like reaction detection unit as a detection substance, and DNA can be detected by a hybridization reaction between the nucleic acid probe and the sample DNA. In this case, if a labeling substance is attached to the detection substance, detection can be performed by detecting the presence or absence of the labeling substance. The sample DNA can be prepared by adjusting DNA extracted from blood or the like by the PCR method, the LAMP method, or the like. In addition, by preparing a plurality of nucleic acids having different sequences as nucleic acid probes that are recognition substances, it is possible to detect the sequence of the sample DNA as the detection substance. In this case, a reagent storage unit may be provided on the substrate to store the detection substance.

  In addition, a PCR reaction unit is provided on the substrate, DNA extracted from blood or the like is continuously amplified on the chip by PCR reaction, and this is used as a sample, and the reaction detection unit determines whether or not there is a reaction with a recognition substance. It may be detected. Specifically, for example, DNA extracted from blood or the like is stored as a detection substance in a well-like reagent storage part, and dispensed to the PCR reaction part by a dispensing operation, and the detection substance adjusted by the PCR reaction is in a well state. It may be dispensed to the reaction detection part. You may send a liquid from a well-like reagent accommodating part to a PCR reaction part and a well-like reaction detection part using a flow path.

  It can also be used to analyze single nucleotide gene polymorphisms (SNPs). Note that there may be a plurality of recognition substances, and when the detection substance is not fluorescently labeled, it is sufficient that one of the recognition substances is labeled.

  In addition, as the labeling substance, a substance that acts specifically on the reacted recognition substance and detection substance can be added after the reaction between the recognition substance and the detection substance. Such an example includes an intercalator for detecting DNA. Further, the labeling substance here includes indirect substances. That is, a substance that binds to a fluorescent substance or the like may be bound to a recognition substance or a detection substance as a labeling substance, and the fluorescent substance may be added later.

Alternatively, SNP or DNA may be detected by performing a multistep reaction.
For example, an invader assay method (Third Wave Technologies, Inc. (Madison, Wisconsin, USA)) may be used, thereby enabling realization of SNP analysis.

  In this case, a plurality of types of recognition substances may be used. At least one type of recognition substance may be placed in advance in the well-like reaction detection unit, and then the detection substance and the recognition substance may be injected simultaneously or sequentially to carry out the reaction.

Further, a solution having a lighter specific gravity than the reaction solution such as mineral oil may be added to the well-like reaction detection unit and the PCR reaction unit in order to prevent the reaction solution from drying.
Further, the recognition substance may be fixed in the well-like reaction detection unit or may be held without being fixed.

  The well-like reaction detection unit, reagent storage unit, and PCR reaction unit may be covered with a lid material such as a film.

The present invention is characterized in that the presence or absence of a reaction in the well-like reaction detector is detected by fluorescence detection from the lower direction of the reaction vessel.
An example is shown in FIG. In FIG. 5, it has the excitation source 10 and the fluorescence detection part 9 in the lower direction of the reaction container 1, and it irradiates the light of the wavelength which can excite a fluorescent substance from the excitation source 9, and is excited by excitation light, The emitted fluorescence is detected by the fluorescence detection unit 9.

Next, a case where the above-described reaction container is actually used for the test will be described.
<Example>
(Chip creation (molding))
The well-shaped detection chip of FIG. 1 is produced by molding. As the resin used for molding, a molded product was prepared using NOVTIC PP MA04A from Nippon Polypro Co., Ltd. The molding temperature was 210 ° C., the clamping force was 50 ton F, the mold temperature was 20 ° C., and the measurement value was 24 mm. The molding machine performs molding with Sumitomo Heavy Industries Psycap M3, and the screw system is φ35 mm. The detection part bottom thickness was 0.3 mm.

(Measurement of transmittance in the well)
When the transmittance in the well was measured using a transmittance measuring device (Olympus OSP-SP200), the transmittance was 84.3% at 470 nm and 85.2% at 520 nm. Further, the transmittance was 86.4% at 570 nm, and 87.4% at 620 nm.
Since it was difficult to measure the haze value in the well, a 0.3 mm plate was prepared by injection molding, and the haze value of the plate was measured using a haze meter (NIPPON DENSHOKU HAZE Meter NDH 2000). The average value obtained by measuring three different points was 5.06 (5.14, 5.08, 4.98).
Further, the center line average roughness at the bottom of the well was measured with an OLYMPUS scanning laser microscope OLS1100. In addition, the average value measured in three places was used. The value obtained was 0.508 μm.

(Invader reaction)
The invader reaction is an invader assay kit (according to Third Wave Technology).
0.15 μl of invader probe was added to the well-like reaction detector and dried.
An invader probe in which 3.644 μl of invader reagent, 0.75 μl of FRET to which FAM is bound as a fluorescent substance, and 0.75 μl of Clevase are dried is dried at the bottom with respect to 1 μl of a PCR product adjusted by PCR reaction as a detection substance. Add 2 μl to each well-like reaction detector. 4 μl of mineral oil is added and an invader reaction is performed at 61 ° C. for 40 minutes. When the reaction was complete, fluorescence detection was performed. In addition, the invader probe, invader reagent, FRET, and Clevase here are recognition substances.

(Fluorescence detection)
Measurement was set at a location 2 mm below the reaction vessel, and detection was performed using real-time PCR (ABI PRISM 7000).
The reaction and fluorescence detection were performed in three wells. The results are shown in FIG.

<Comparative example>
(Chip creation (molding))
The well-shaped detection chip of FIG. 1 is produced by molding. As the resin used for molding, a molded product was prepared using NOVTIC PP MA04A from Nippon Polypro Co., Ltd. The molding temperature was 210 ° C., the clamping force was 50 ton F, the mold temperature was 20 ° C., and the measurement value was 24 mm. The molding machine performs molding with Sumitomo Heavy Industries Psycap M3, and the screw system is φ35 mm. The thickness of the bottom thickness of the detection part was the same as that in the figure, but the detection part thickness was 1.0 mm.

(Measurement of transmittance in the well)
When the transmittance in the well was measured in the same manner as in Example, the transmittance was 72.1% at 470 nm and 75.7% at 520 nm. Further, the transmittance was 78.8% at 570 nm and 80.9% at 620 nm.
Since it was difficult to measure the haze value in the well, a 1.0 mm plate was prepared by injection molding, and the haze value of the plate was measured using a haze meter (NIPPON DENSHOKU HAZE Meter NDH 2000). The average value of three different points measured was 11.89 (11.94, 12.83, 10.91).
Further, the center line average roughness at the bottom of the well was measured with an OLYMPUS scanning laser microscope OLS1100. In addition, the average value measured in three places was used. The value obtained was 0.579 μm.

(Invader reaction)
The invader reaction is an invader assay kit (according to Third Wave Technology).
0.15 μl of invader probe was added to the well-like reaction detector and dried.
An invader probe in which 3.644 μl of invader reagent, 0.75 μl of FRET to which FAM is bound as a fluorescent substance, and 0.75 μl of Clevase are dried is dried at the bottom with respect to 1 μl of a PCR product adjusted by PCR reaction as a detection substance. Add 2 μl to each well-like reaction detector. 4 μl of mineral oil is added and an invader reaction is performed at 61 ° C. for 40 minutes. When the reaction was complete, fluorescence detection was performed. In addition, the invader probe, invader reagent, FRET, and Clevase here are recognition substances.

(Fluorescence detection)
Measurement was set at a location 2 mm below the reaction vessel, and detection was performed using real-time PCR (ABI PRISM 7000).
The reaction and fluorescence detection were performed in three wells. The results are shown in FIG.

<Evaluation>
As shown in FIGS. 6 and 7, the strength of the sample of the example became constant from about 18 minutes, and the strength was also high. On the other hand, the strength of the sample of the comparative example was not constant even after 20 minutes, it was difficult to determine, and the strength that finally became constant was low. Further, all the three samples of the examples obtained data having no variation, but the samples of the comparative example showed some variation.
Also, when analyzing using the time until the intensity becomes constant and the slope of the graph, the comparative example is more difficult to determine than the example.

It is the schematic which shows an example of the reaction container of this invention. It is the schematic which shows an example of the reaction container of this invention. It is the schematic which shows an example of the reaction container of this invention. It is the schematic which shows an example of the reaction container of this invention. It is the schematic explaining the fluorescence detection using the reaction container of this invention. It is a graph which shows the result of the fluorescence measurement of an Example. It is a graph which shows the result of the fluorescence measurement of a comparative example.

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 Reaction container 2 Substrate 3 Well-like reaction detection part 4 Distance between well-like reaction detection part bottom and substrate back surface 5 Reagent storage part 6 PCR reaction part (channel type)
7 Channel 8 Reaction solution 9 Fluorescence detector 10 Excitation source

Claims (9)

  In a reaction vessel having a well-like reaction detection unit on a substrate, the average light transmittance at a wavelength of 400 to 650 nm between the bottom surface of the well-like reaction detection unit and the back surface of the substrate is 80% or more, and the haze is 10 A reaction vessel characterized by being in the following range.   The reaction container according to claim 1, wherein a distance between the bottom surface of the well-like reaction detection unit and the back surface of the substrate is 0.3 or more and less than 1 mm.   The reaction container according to claim 1, wherein a bottom surface of the well-like reaction detection unit is flat.   The reaction container according to claim 1, wherein there are a plurality of well-like reaction detection units.   The reaction container according to claim 1, wherein the base material is a cycloolefin resin or a methylpentene resin.   Furthermore, it has a well-shaped reagent storage part, The reaction container in any one of Claims 1-5 characterized by the above-mentioned.   Furthermore, it has a PCR reaction part, The reaction container in any one of Claims 1-6 characterized by the above-mentioned.   The substrate has a well-like reaction detection portion, and the average light transmittance at a wavelength of 400 to 650 nm between the bottom surface of the well-like reaction portion and the back surface of the substrate is 80% or more, and the haze is 10 or less. A step of injecting a recognition substance into the well-like reaction detection unit; and a step of injecting a detection substance into the well-like reaction detection unit, wherein either the recognition substance or the detection substance is fluorescently labeled, and the recognition A method for detecting a substance, comprising the step of detecting fluorescence from the bottom side of the well-like reaction detection unit by the presence or absence of a reaction between the substance and the detection substance.   The method for detecting a substance according to claim 7, wherein the detection substance is a nucleic acid.