JP2005292051A - Carrier, substance detection device, manufacturing method thereof, and detection method - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for easily reducing the background noise, when detecting the radiation light emitted when a carrier is irradiated with an irradiation light, and when a substance is carried for detecting the substance carried by the carrier. <P>SOLUTION: The carrier for carrying the substance to be detected is dyed with ink. The refractive index of the carrier is changed by clearing, or the like prior to detection. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to a carrier dyed with ink, a substance detection device having the carrier, a manufacturing method thereof, and a detection method for detecting a substance carried on the carrier.

  Macroarrays and microarrays are known as devices in which DNA, RNA, and proteins are fixed in an array on a carrier. These arrays are used for research and diagnosis in the fields of biology and medicine.

  DNA, RNA, proteins, etc. are fixed on the surface of the array. Examples of immobilization methods include those conventionally used in Southern blotting, Northern blotting, Western blotting, DNA chip methods for direct DNA synthesis on a substrate, DNA clones and DNA amplified by PCR, There is a DNA microarray method in which synthesized DNA or the like is aligned and fixed on a substrate using an apparatus called a spotter. The protein can be immobilized on a substrate using a spotter as in the DNA microarray method.

  In general, target molecules labeled with RI (radioisotope) or fluorescent dyes are used to detect target molecules that bind to the probes on these arrays. In addition, an RLS assay system (Qiagen) has been developed that has the same detection sensitivity as RI and has a very low risk. This method uses target DNA labeled with fluorescein or biotin, etc., and light scattering particles (RLS particle (trademark)) combined with anti-fluorescein antibody or anti-biotin antibody that recognizes the target DNA. Scattered light obtained from the light scattering particles is detected.

  Macroarrays and microarrays differ in the size of the carrier, the density of the substance carried, and the overall size. For the macroarray, a membrane such as nitrocellulose, nylon, PVDF, or plastic is used, and for the microarray, a substrate such as glass or silicon is used. Glass slides coated with nitrocellulose, such as Fast (registered trademark; Schleicher & Schuell) -slide, are also used, but most of the fluorescence detection systems used in microarrays have a high background. not being used. However, the amount and fixing force of the substance supported on the surface coated with the membrane are remarkably improved as compared with those of the substrate.

Polyolefin is used as a support for such a DNA microarray, and it has been disclosed that it is black or transparent to lower the background (see, for example, Patent Document 1).
Japanese Patent Application Laid-Open No. 2003-28867

  However, it is usually not easy for an experimenter using a commercially available carrier to use a polyolefin as a carrier for DNA or protein and make the carrier black or transparent. That is, until now, there has been no method for the experimenter to easily reduce the background noise regardless of the type of carrier.

  Therefore, the present invention can easily reduce background noise, a carrier dyed with ink, a substance detection device having the carrier, a manufacturing method thereof, and a background noise can be easily reduced. It is an object of the present invention to provide a method for detecting a substance to be detected carried on a carrier, which can be carried out.

  The carrier of the present invention is a carrier for carrying a substance to be detected and is characterized by being dyed with ink. Here, the ink refers to a colored liquid and is mainly used for writing or printing, but the original use may not be for writing or printing. The carrier preferably contains nitrocellulose as a material, but other materials may be used.

  Further, in order to detect the substance carried on the carrier, irradiation light may be applied to the carrier and radiation emitted when the substance is carried may be detected. The emitted light may be fluorescence or resonance scattered light.

  The carrier may be dyed with black ink or ink having a characteristic of absorbing light having the wavelength of the irradiation light.

  The carrier may carry a substance to be detected.

  Further, all the substances to be detected may be DNA.

  Furthermore, the substance detection instrument of the present invention is a substance detection instrument having a carrier for carrying a substance to be detected, wherein the carrier is dyed with ink. The carrier may contain nitrocellulose as a material.

  Further, in the substance detection instrument, in order to detect the substance carried on the carrier, irradiation light is applied to the carrier, and radiation emitted when the substance is carried is emitted. It may be detected. The emitted light may be fluorescence or resonance scattered light.

  In the substance detection instrument, the ink may be black ink or ink having a characteristic of absorbing light having the wavelength of the irradiation light.

  The substance may be DNA.

  Furthermore, the detection method of the present invention is a detection method for detecting a substance carried on a carrier, and includes a step of irradiating the carrier with irradiation light, and radiation emitted when the substance is present. Detecting, and the carrier is dyed with ink. The carrier may contain nitrocellulose as a material. Further, the emitted light may be fluorescence or resonance scattered light. The carrier may be dyed with black ink or ink having a characteristic of absorbing light having the wavelength of the irradiation light. The substance may be DNA. Note that this detection method may further include a step of performing a process of changing the refractive index of the carrier.

  Furthermore, the method for producing a substance detection instrument of the present invention is a method for producing a substance detection instrument having a carrier for carrying a substance to be detected, and includes a step of dyeing the carrier with ink. It is characterized by. The carrier may contain nitrocellulose as a material. The carrier may be dyed with black ink. Alternatively, in order to detect the substance carried by the carrier, it is used to irradiate the carrier with irradiation light and detect radiation emitted when the substance is carried on the carrier. In the method for manufacturing a substance detection instrument having a carrier, the carrier may be dyed with an ink having a characteristic of absorbing light having a wavelength of the irradiation light.

  Furthermore, the detection method of the present invention detects a substance carried on the carrier by irradiating the carrier with irradiation light and detecting radiation emitted when the substance is carried on the carrier. In the detection method, the carrier is an artificial material, and a process of changing a refractive index of the carrier is performed.

  In this detection method, the support may be a membrane made of nitrocellulose, nylon, PVDF, or a mixture thereof. Further, the refractive index of the carrier may be changed by making the carrier transparent. In this case, the angle of the irradiated light may be changed or the angle of the emitted light may be changed by changing the refractive index of the carrier. In addition, you may change the refractive index of the said support body by using the tissue specimen mounting agent.

  According to the present invention, it is possible to easily reduce background noise, a carrier stained with ink, a substance detection device having the carrier, a method for manufacturing the same, and a background noise can be easily reduced. It is possible to provide a method for detecting a substance to be detected carried on a carrier, which can be carried out.

  A detection method for detecting a substance carried on a carrier having a substance detection instrument of the present invention includes a step of irradiating the carrier with irradiation light and detecting radiation emitted when the substance is present. . Hereinafter, a method for producing a carrier and a substance detection device and a method for detecting a substance to be detected will be described in detail together with examples.

  The objects, features, advantages, and ideas of the present invention will be apparent to those skilled in the art from the description of the present specification, and those skilled in the art can easily reproduce the present invention from the description of the present specification. it can. The embodiments and specific examples of the invention described below show preferred embodiments of the present invention and are shown for illustration or explanation, and the present invention is not limited to them. It is not limited. It will be apparent to those skilled in the art that various modifications and variations can be made based on the description of the present specification within the spirit and scope of the present invention disclosed herein.

== Method for producing carrier and substance detection instrument ==
Substances to be detected include, for example, oligonucleotides synthesized in vitro, DNA such as cDNA and BAC, RNA such as isolated mRNA and RNA synthesized in vitro, peptides synthesized in vitro, and tissue extracts Proteins such as proteins and antibodies are preferred, but the present invention is not limited to these as long as the substance is supported on a carrier and can be detected by the above detection method. Note that the form of the specimen containing this substance may be liquid such as a DNA solution, or may be solid such as a tissue section. Further, it may be an organic material such as agarose including agarose or a polymer including polyacrylamide, an inorganic material such as silicon or metal, a colloidal shape, a gel shape, a semi-solid shape, or the like.

  There are no particular limitations on the carrier possessed by the substance detection device as long as it can carry the substance to be detected as described above, and can detect the substance by the detection method described above and can be stained with ink. The material of the carrier carrying the substance to be detected is preferably nitrocellulose, but may be nylon or plastic, and is not particularly limited. The form of the carrier may be a membrane or may be a glass slide or substrate coated, but is not limited thereto. For example, representative membranes include, but are not limited to, conventional blotting such as Southern blotting, Northern blotting, Western blotting, and nitrocellulose membranes and nylon membranes used for macroarrays. Examples of the support coated on the slide glass or the substrate include nitrocellulose coated on Fast (registered trademark; Schleicher & Schuell) -slide.

  When these carriers are used, they are dyed with ink in order to reduce background noise. The ink is preferably a black ink, but may be any color ink having a characteristic of absorbing light having a wavelength of irradiation light used for detecting a substance. Thereby, it is possible to prevent the background noise caused by the carrier from being generated by detecting the irradiation light reflected by the carrier together with the emitted light. The original use of this ink is not limited to writing and printing, but it is preferably a ballpoint pen ink. In order to dye the carrier with ink, for example, the carrier may be immersed in ink for a predetermined time, and then the dyed density of the carrier can be adjusted by washing the dyed carrier with water. By using ink for dyeing in this way, it becomes possible to dye the carrier on the side of the experimenter detecting the substance, so that the carrier of the present invention can be easily produced.

  As a specific method for supporting the substance to be detected on the carrier, for example, DNA may be synthesized on the carrier or the synthesized DNA may be placed on the carrier. It may be fixed by covalently bonding to the surface of the support using an agent or by baking the support in a vacuum oven. In the case of a membrane, it can be crosslinked with an alkaline solution or baked in a vacuum oven. In addition, in the case of proteins, cysteine may be added to the terminal and then fixed to the surface of the support by a thiol coupling method, but may be fixed only by blotting. In addition, the carrying method and the fixing method are not limited to these, and an appropriate one for the substance to be detected is selected.

  Substance detection devices equipped with these supports include, for example, glass slides coated with gelatin and silica, glass slides coated with nitrocellulose such as Fast-slide, Although there exists a board | substrate etc. which a cellulose membrane was affixed, the instrument for substance detection of this invention is not limited to these.

  In view of the above, examples of specific forms of a substance detection instrument carrying a substance to be detected include a slide glass on which a tissue section is pasted, a nitrocellulose membrane on which nucleic acids and proteins are blotted, and various types of oligos Various forms such as a DNA array having nucleotides and BACs, and a protein microarray to which other types of antibodies are immobilized are conceivable, but the substance detection instrument of the present invention is not particularly limited thereto.

== Detection method of substances to be detected ==
As a specific method for detecting the substance carried on the carrier having the substance detection instrument of the present invention, an appropriate method is selected for the substance to be detected. Typically, a detection means labeled with a label such as fluorescence is administered to bind to the substance to be detected, the support is irradiated with irradiation light, and the emitted radiation is detected.

  As the detection means, for example, when the detection target is a nucleic acid such as single-stranded DNA or RNA, DNA or RNA having a complementary strand sequence can be used. When the detection target is a protein, the protein When the detection target is an antibody, a protein that is recognized by the antibody can be used. Actually, a substance that does not bind to a detection target such as DNA, RNA, or protein may be mixed in the detection means. For example, an extract from a tissue or a crudely purified nucleic acid may be used as the detection means. . In this way, an appropriate detection means is selected according to the substance to be detected in consideration of the experimental purpose.

  As the primary label for directly labeling the detection means, for example, a fluorescent label, a biotin label, and other tags can be used. In the case of labeling with a fluorescent label, the detection means bound to the substance to be detected is detected by irradiating the support with excitation light and detecting the emitted light. In any case, the detection means indirectly bound to the substance to be detected by detecting the primary label using the primary label-specific antibody labeled with fluorescence or light scattering particles as the secondary label. May be detected. When the secondary label is a light scattering particle, the carrier is irradiated with white light, and the substance to be detected is detected by detecting the resonance scattered light. In addition, signals can be amplified using immunochemical techniques such as the ABC method.

  The emitted light emitted by the detection method as described above can be detected and analyzed by a fluorescence detection device such as a DNA microarray scanner in the case of fluorescence, and by a HiLight array detection system (Qiagen) in the case of resonance scattered light.

== Adjustment of refractive index of carrier ==
When observing biological tissue with a microscope, the internal structure cannot be observed because the tissue is translucent. Therefore, xylene and the like are replaced with water in the tissue to be transparent, and further replaced with a tissue specimen mounting agent to maintain transparency.

  On the other hand, in the detection method for detecting the substance carried on the carrier, the radiation is emitted when the carrier is irradiated with the irradiation light and the substance is carried on the carrier. Reflection may be detected as background noise. At this time, the occurrence of background noise can be suppressed by performing a process of changing the refractive index of the carrier.

  The carrier used here may be an artificial material, and examples thereof include membranes, substrates, plastics, acrylamide gels, and the like, but are not limited thereto. In order to change the refractive index of the carrier, the carrier may be transparent, but it is not necessarily transparent. The method of changing the refractive index of the carrier can be made transparent by using xylene or a tissue specimen mounting agent, but the drug is limited to these if the carrier can be made transparent and the state can be maintained. In addition, a plurality of reagents may be used in combination.

  In the following examples, the support was made transparent using a tissue specimen mounting medium. Here, biolite (manufacturer: Koken Co., Ltd., distributor: Oken Shoji Co., Ltd., trademark) was used as a tissue specimen mounting medium. Note that the tissue specimen mounting agent also has an effect of preventing the fixation of the carrier and the fading of the pigment.

  When QIAGEN's RLS assay system is used for the membrane, the reflected light is so strong that it affects the background and cannot be detected. Encapsulants can be used to make the membrane transparent, thereby reducing background noise.

  In this example, a method for easily reducing background noise when analyzing a DNA microarray will be described in detail.

== Preparation of stained FAST-slide ==
FAST-slide was quickly immersed in PIN101 ink manufactured by Mitsubishi Pencil Co., Ltd. and allowed to stand for about 5 minutes. The FAST-slide was taken out, quickly put into water, and washed with running water for about 5 minutes. The washed FAST-slide was centrifuged at 2000 rpm for 5 minutes with a plate centrifuge to remove and dry the water, and then dried with a desiccator and stored as it was. As a control, unstained FAST-slide was used.

== Preparation of DNA microarray ==
4 μl of 1 μg / μl BAC DNA was placed in a 0.5 ml microcentrifuge tube and cut by applying ultrasonic waves for about 1 hour in a bathtub-type ultrasonic cleaner. 1 μl of 5 × denaturing buffer was added and pipetted with pipetman about 20 times to denature the DNA. BAC DNA was spotted on FAST-slide (humidity 60%) using a DNA microarray production apparatus (QArray manufactured by Genetix). The FAST-slide was heated in a vacuum oven at 80 ° C. for 30 minutes, then washed by putting it in and out of the neutralization buffer about 100 times, and further washed with running water. After centrifuging at 2000 rpm for 5 minutes with a plate centrifuge to remove and dry the moisture, the plate was dried with a desiccator and stored as it was.

== Pre-hybridization ==
10 μl of 10 μg / μl salmon sperm single-stranded DNA was added to 600 μl of 1 × MicroHybridization Prehyb / Wash Buffer attached to the FAST-slide kit, added dropwise to the DNA microarray, and 37 ° C. using the Hybridization Chamber attached to the kit. Shake for hours.

== CGH hybridization to fluorescence imaging and analysis thereof ==
1 μg of human genomic DNA labeled with Cy3 or Cy5 and 30 μl of Cot-1 DNA were mixed, ethanol precipitated, suspended in 600 μl of Hybridization Buffer attached to FAST-slide, treated at 100 ° C. for 5 minutes, and incubated at 37 ° C. for 120 minutes.

  This hybridization solution was replaced with a pre-hybridization solution on a DNA microarray, and incubated at 37 ° C. for 12 hours or more using a hybridization chamber.

  After completion of hybridization, the DNA microarray was washed twice at 42 ° C. for 20 minutes using 1 × MicroHybridization Prehyb / Wash Buffer, and then washed once at room temperature for 5 minutes using 1 × SSC. After centrifuging with a plate centrifuge at 2000 rpm for 5 minutes to remove moisture and drying, it was dried again in a 60 ° C. incubator for 5 minutes. Thereafter, FAST-slide was made transparent using biolite according to the method described later.

  The DNA microarray was scanned using a DNA microarray scanner (ScanArray5000 manufactured by Packerd) (Focus: about -1986, Laser Power: 100%, PMT: about 50%).

  1 and 2 show the results of using unprocessed FAST-slide, and human genomic DNAs labeled with Cy3 and Cy5, respectively, were used as detection targets. FIG. 5 shows the results of dot plot analysis in which the signal intensity of Cy3 is plotted on the x-axis and the signal intensity of Cy5 is plotted on the y-axis for each DNA sample. FIG. 7 shows an enlarged view of the analyzable region (region in which the dot value is larger than the background noise value) in FIG.

  3 and 4 show the results of using FAST-slide stained with black ink, and human genomic DNAs labeled with Cy3 and Cy5, respectively, were used as detection targets, as in FIGS. FIG. 6 shows the results of dot plot analysis when using stained FAST-slide. FIG. 8 shows an enlarged view of the analyzable region of FIG.

  1 and 3 are compared for the experiment using Cy3, and FIGS. 2 and 4 are compared for the experiment using Cy5. In both cases, the stained FAST-slide was used at first glance. It can be seen that the signal-to-noise ratio increases with time, and that many signals can be detected. When the S / N ratio was compared with the actually measured signal intensity, the signal was weak in the analyzable region, and the average increased 1.94 times when using Cy3 and 1.23 times when using Cy5. Similarly, the signal-to-noise ratio increased in an area where the signal was strong in the analyzable region, with an average of 2.00 times when using Cy3 and an average of 5.27 times when using Cy5.

  Further, comparing FIG. 7 and FIG. 8, it became possible to detect a trace amount of signal by using stained FAST-slide, and the number of samples that could be analyzed increased from 242 clones to 582 clones.

== CGH hybridization using RLS assay system-analysis ==
1 μg of human genomic DNA labeled with biotin or fluorescein and 30 μl of Cot-1 DNA were mixed, ethanol precipitated, suspended in 600 μl of Hybridization Buffer attached to FAST-slide, treated at 100 ° C. for 5 minutes, and incubated at 37 ° C. for 120 minutes.

  This hybridization solution was replaced with a pre-hybridization solution on a DNA microarray, and incubated at 37 ° C. for 12 hours or more using a hybridization chamber.

  After completion of hybridization, the DNA microarray was washed twice at 42 ° C. for 20 minutes using 1 × MicroHybridization Prehyb / Wash Buffer, and then washed once at room temperature for 5 minutes using 1 × SSC. After centrifuging with a plate centrifuge at 2000 rpm for 5 minutes to remove moisture and drying, it was dried again in a 60 ° C. incubator for 5 minutes.

 Below, using the RIA method manufactured by QIAGEN, labeled FAST-slide with light scattering particles according to the protocol provided by QIAGEN, then transparentized FAST-slide using biolite according to the method described below, and detected the signal with a dedicated scanner. . FIG. 9 shows the result.

== Transparency of carrier ==
First, the labeled DNA microarray was dried, and 300 μl of violite was placed on the DNA microarray. Before the biolite was dried, the biolite was stretched with a cover glass so that bubbles did not enter, and was covered with the cover glass. After waiting for the biolite to dry for about 30 minutes at room temperature, the biolite protruding from the slide glass was scraped off, and a signal was detected with a detection scanner.

 FIG. 10 shows the signal obtained before clearing (right of FIG. 10) and the signal obtained after clearing for FAST-slide labeled using the RLS assay system after CGH hybridization. The comparison of (FIG. 10 left) is shown. Prior to clearing, the background noise was about 24,000, whereas after clearing, the background noise was reduced to about 15000.

In one Example using this invention, it is a figure which shows the signal obtained when using unprocessed FAST-slide and using human genomic DNA labeled with Cy3 for hybridization. In one Example using this invention, it is a figure which shows the signal obtained when using unprocessed FAST-slide and using human genomic DNA labeled with Cy5 for hybridization. In one Example using this invention, it is a figure which shows the signal obtained when using dyed FAST-slide and using human genomic DNA labeled with Cy3 for hybridization. In one Example using this invention, it is a figure which shows the signal obtained when dye | staining FAST-slide and using human genomic DNA labeled with Cy5 for hybridization. The results of dot plot analysis of the signals obtained in FIGS. 1 and 2 are shown. The result of dot plot analysis of the signals obtained in FIGS. 3 and 4 is shown. FIG. 6 shows an enlarged view of the analyzable region in FIG. 5. The enlarged view of the analysis possible area | region of FIG. 6 is shown. In one Example using the present invention, a diagram showing a signal obtained by reacting an anti-biotin antibody-gold particle complex with a stained human FAST-slide and using human genomic DNA labeled with biotin for hybridization. It is. In one example using the present invention, a stained FAST-slide was used, and biotin-labeled human genomic DNA was used for hybridization to react with an anti-biotin antibody-gold particle complex, and then transparent using biolite. It is the figure which compared the signal obtained before making it into left (left figure) and after making transparent (right figure).

Claims (30)

A carrier for carrying a substance to be detected,
A carrier characterized by being dyed with ink.   The carrier according to claim 1, wherein the carrier contains nitrocellulose as a material.   In order to detect the substance carried on the carrier, irradiation light is applied to the carrier and radiation emitted when the substance is carried is detected. Item 3. The carrier according to Item 1 or 2.   The carrier according to claim 3, wherein the emitted light is fluorescence or resonance scattered light.   The carrier according to any one of claims 1 to 4, which is dyed with black ink.   The carrier according to claim 3 or 4, wherein the carrier is dyed with an ink having a characteristic of absorbing light having a wavelength of the irradiation light.   The carrier according to any one of claims 1 to 6, wherein the substance to be detected is carried.   The carrier according to any one of claims 1 to 7, wherein the substance is DNA.   A substance detection instrument having a carrier for carrying a substance to be detected, wherein the carrier is dyed with ink.   The instrument for substance detection according to claim 9, wherein the carrier includes nitrocellulose as a material.   In order to detect the substance carried on the carrier, irradiation light is applied to the carrier and radiation emitted when the substance is carried is detected. Item 9. The substance detection instrument according to Item 9 or 10.   The instrument for detecting a substance according to claim 11, wherein the emitted light is fluorescence or resonance scattered light.   13. The substance detection instrument according to claim 9, wherein the ink is black ink.   The substance detection instrument according to claim 11 or 12, wherein the ink is an ink having a characteristic of absorbing light having a wavelength of the irradiation light.   The instrument for detecting a substance according to any one of claims 9 to 14, wherein the substance is DNA. A detection method for detecting a substance carried on a carrier,
Irradiating the carrier with irradiation light;
Detecting radiation emitted in the presence of the substance;
Including
A detection method, wherein the carrier is dyed with ink.   The detection method according to claim 16, wherein the carrier includes nitrocellulose as a material.  The detection method according to claim 16 or 17, wherein the emitted light is fluorescence or resonance scattered light.   The detection method according to claim 16, wherein the carrier is dyed with black ink.   The detection method according to claim 16, wherein the carrier is dyed with an ink having a characteristic of absorbing light having a wavelength of the irradiation light.   The detection method according to claim 16, wherein the substance is DNA. A method for producing a substance detection instrument having a carrier for carrying a substance to be detected,
A method for producing a substance detection instrument, comprising the step of dyeing the carrier with ink.   The method for manufacturing a substance detection instrument according to claim 22, wherein the carrier includes nitrocellulose as a material.   The method for manufacturing a substance detection instrument according to claim 22 or 23, wherein the carrier is dyed with black ink. In order to detect the substance carried on the carrier, it is used to irradiate the carrier with irradiation light and detect radiation emitted when the substance is carried on the carrier. A method for producing a substance detection instrument according to claim 22 or 23, comprising a carrier to be obtained,
A method for producing a substance detection instrument, comprising the step of dyeing the carrier with ink having a characteristic of absorbing light having a wavelength of the irradiation light. In a detection method for detecting a substance carried on a carrier, irradiating the carrier with irradiation light and detecting radiation emitted when the substance is carried on the carrier,
The carrier is made of an artificial material,
The detection method characterized by performing the process which changes the refractive index of the said support body.   27. The detection method according to claim 26, wherein the carrier is a membrane made of nitrocellulose, nylon, PVDF, or a mixture thereof.   28. The detection method according to claim 26 or 27, wherein the refractive index of the carrier is changed by making the carrier transparent.   28. The detection method according to claim 26 or 27, wherein a refractive index of the carrier is changed by using a tissue specimen mounting agent. The detection method according to any one of claims 16 to 21, further comprising a step of performing a process of changing a refractive index of the carrier.