JP4671548B2 - Analyte measurement method and automatic gene detection apparatus - Google Patents

Description

[0001]
BACKGROUND OF THE INVENTION
  The present invention provides a simple and highly sensitive method for detecting an analyte in a solution.LawYoDressRelated to the position. More specifically, the present invention relates to a method for specifically detecting whether or not a target nucleic acid is present in a solution.LawYoRuinsThe present invention relates to a gene detection device. The method and device of the present invention can be integrated at high density, and can be applied to screening of drugs such as therapeutic agents, gene fingerprinting, and determination of gene base sequence (SBH: Sequencing By Hybridization). It is.
[0002]
[Prior art]
The total number of genes present in an organism is said to be about 6,000 in yeast and about 100,000 in humans. Sequencing of human genes is almost complete, and more than 80% of human genes are part of them as EST (Expressed Sequence Tag: 200-300 base sequence read from the end of cDNA) It is said that the sequence has already been published.
[0003]
In general, gene detection methods such as genotype analysis for each cell or tissue, comparison of gene expression patterns, mapping of genetic mutation polymorphisms (diagnosis), analysis of functional base sequences present in the genome, etc. denatured into single strands The presence of the target gene is confirmed by detecting a nucleic acid probe specifically hybridized to the gene sample. Recently, DNA chips have been employed in such gene detection methods. For example, Hyseq discloses a nucleotide sequence analysis method using a DNA chip (see US Pat. No. 5,525,464).
[0004]
The DNA chip is said to be able to analyze all genes contained in a living body in about 1 to 2 days. Since the base sequence can be easily and quickly analyzed, the development of utilization technology of the DNA chip is rapidly progressing. It is out. However, the problem with determining unknown sequences using a DNA chip is that the DNA chip gives a signal equivalent to a perfect match for several terminal mismatches between the gene and the nucleic acid probe. Has been pointed out. Therefore, it is necessary to technically study a method for accurately discriminating between the terminal mismatch and the complete match.
[0005]
A typical gene analysis method of the prior art including a DNA chip will be described. First, a gene is extracted from a biological sample and, if necessary, cleaved with an appropriate restriction enzyme, followed by electrophoresis and Southern blotting. Next, a nucleic acid probe having a base sequence complementary to the target gene is labeled with a radioisotope or a fluorescent dye, and hybridized with the blotted gene. Next, the hybridized nucleic acid is exposed to an X-ray film at a low temperature, and then a RI scanner is used, or fluorescence emission generated by irradiating a laser is used, or a photodetector such as a high-sensitivity CCD camera. By detecting a fluorescently labeled substance using a photomultiplier tube, the hybridized nucleic acid probe is detected, thereby confirming the presence of the target gene.
[0006]
However, in the detection method using the radioisotope, the radioisotope is used, so the diagnostic place is limited, and the handling of the reagent must be taken care of. In addition, it takes a long time to detect a gene, and the measurement operation is also complicated and complicated. The operation must also be performed by a qualified operator. In addition to this, attention must be paid to waste disposal.
[0007]
In order to improve this point, the development of safe labeling agents to replace radioisotopes is underway. For example, several probes such as a method using an avidin-biotin bond and a method using an enzyme or a fluorescent substance are used. A detection method has already been proposed. A scanner used in such a detection method has a capability of detecting a size of about several tens of microns, and can quantitatively identify spots having an interval of about 100 microns. However, such as when using a laser with two wavelengths, many, and five wavelengths, it cannot sufficiently handle a large number of labels, and cannot scan a wide range at high speed. For example, a scanner using a confocal laser has a relatively low resolution of 5 to 50 μm, which is an obstacle to high-density integration of the probe detection method as described above. In addition, there is a problem that it is impossible to evaluate whether the fluorescence is actually generated by the binding that matches the target sequence, or whether the signal is a non-specific false positive signal that occurs due to the binding in a mismatched state. .
[0008]
As another detection method, there is a mass spectrometric detection method using MALDI-TOF (Matrix-assisted laser deformation Time of Flight) (for example, DNA Mass Array of Sequenom). This TOF mass analyzer ionizes a sample, separates sample molecules according to the mass / electricity ratio using a magnetic field type or quadrupole type device, and measures the time required for the generated ions to make one round of the detector To do. Using a TOF mass analyzer, for example, a primer extension reaction is performed on a fragment of a gene of interest bound to a DNA chip, thereby generating a ladder of bound complementary DNA sequences, and the mass of the DNA with an accuracy of 1 to 2 daltons. Can be detected. However, in principle, the actual analysis time is very short, but on the other hand, it is necessary to perform complicated sample preparation work before mass analysis of a biological sample. take time. In addition, the currently introduced system is large and it takes time to prepare the apparatus.
[0009]
Japanese Patent Laid-Open No. 10-23900 discloses a method and apparatus for detecting a DNA hybridization reaction without labeling. This device includes a DNA separation / extraction module, amplification module, and detection module, and detects changes in phase difference due to double strand formation caused by hybridization between the probe on the chip and denatured single-stranded DNA in the sample. To do.
[0010]
In recent years, a scanning probe microscope (SPM) typified by a scanning tunneling microscope (STM) using an ultrafine probe has been developed. Using this, a scanning probe microscope (SPM) of the atomic order or nanometer order has been developed. Surface observation, atomic manipulation, and surface modification have been actively performed. In particular, from the viewpoint of data storage application, surface modification and surface modification of a storage medium are performed, and a nanometer-sized uneven structure is produced. By using a scanning probe microscope, it is possible to control not only the geometric shape of the sample surface but also physical properties such as magnetic force distribution, surface potential, and optical information of the sample with a resolution of nanometer order. This can be achieved by controlling the voltage applied between the probe and the sample, the accompanying electron injection, heat, and micro force in the nanometer region.
[0011]
Among them, 1989 Stanford University's C.I. F. The group of Prof. Ouate et al. Showed the possibility of ultra-high density recording by forming nanometer-order pit structures on the graphite surface by current heating with STM. With this as a trigger, researches are now actively underway to apply such microfabrication technology to future storage technologies worldwide. For example, in 1990, Mamin et al. Showed dot recording technology by gold electric field evaporation using STM, Betzig et al. Showed magneto-optical recording by a 1992 near-field optical microscope SNOM, and Sato et al. A change record was shown, and Hosoki et al. Verified the possibility of atomic storage by atomic manipulation.
[0012]
In addition, M.M. Despond et al. Despond et al., “VLSI-NEMS Chip for AFM Data Storage”, IEEE Micro Electro Mechanical Systems Technics 1999, pp 564-569, improved wear resistance of SPM probe (probe) Thus, the AFM probes equipped with the deflection detection sensors are arranged in a 32 × 32 matrix on the same plane, and the heated probes are pressed against a recording medium made of PMMA resin to form depressions and detect irregularities. Thus, a prototype of a data storage device for recording and reproducing data was shown. The data mark size recorded by this method is about 40 nm and is 400 GB / inch.²Corresponds to the recording density of.
[0013]
  Others include Vettiger, P. et al. Despont, M .; Drechsler, U .; Durig, U .; Haberle, W .; Lutwyche, M .; I. Rothuzen, H .; E. Stutz, R .; Widmer, R .; Binnig, G .; K. “Millipede” more than one tips and futures AFM data storage, IBM Journal of Research and Development, Vol. 44. Issue 3 323-340, 2000, etc. However, as far as the present inventors know, there is no prior art in which a scanning probe microscope is combined with a DNA chip and applied to a gene analysis method.Other prior art documents include Japanese Patent Laid-Open No. 02-242144 (particularly FIG. 2 and its description), and C.I. A. J. et al. Putman et. al. “Detection of In Situ Hybridization to Human Chromosomes With the Atomic Force Microscope”, Cytometry 14: 356-3
61 (1993).
[0014]
[Problems to be solved by the invention]
  The present invention solves the above-mentioned problems of the prior art, accumulates analytes in a solution at high density, and can detect them simply and with high sensitivity.LawYoDressThe purpose is to provide a device.
[0015]
[Means for Solving the Problems]
  The present invention relates to an analyte measurement method, which includes capturing an analyte of interest in at least one region on a substrate, modifying a geometric shape of the region in which the analyte is captured, and modifying Detecting the generated geometric shape, thereby detecting the presence of the analyte.
  More specifically, the present invention can relate to the following items.
  (Item 1)
  Capturing the target analyte in the nucleic acid probe on a substrate having a support layer, a photoresist layer disposed on the support layer, and a nucleic acid probe fixed to the support layer on the surface;
  Applying energy selected from the group consisting of visible light, ultraviolet light, infrared light, X-rays, and electron beams to the surface of the substrate, modifying the photoresist layer in the region where the analyte is captured, Forming a concavo-convex shape on the substrate, and
  A method for measuring an analyte, comprising: detecting the uneven shape with a scanning probe microscope, thereby detecting the presence of the analyte.
  (Item 2)
  2. The analyte measuring method according to item 1, wherein the step of forming a concavo-convex shape on the substrate is performed in the presence of a labeling substance that specifically binds to the analyte captured on the substrate.
  (Item 3)
  2. The analyte measurement method according to item 1, wherein the analyte has a labeling substance.
  (Item 4)
  Item 2. The analyte measurement method according to Item 1, wherein the analyte has a labeling substance made of a light shielding substance.
  (Item 5)
  2. The method for measuring an analyte according to item 1, wherein the analyte is a nucleic acid.
  (Item 6)
  2. The analyte measuring method according to item 1, further comprising a step of fixing the uneven shape of the substrate.
  (Item 7)
  2. The method for measuring an analyte according to item 1, further comprising a step of etching the support layer using the photoresist layer as a mask after the step of forming an uneven shape on the substrate.
[0040]
DETAILED DESCRIPTION OF THE INVENTION
In the following, embodiments of the present invention in which the target analyte is a nucleic acid, particularly DNA, will be described, but these are examples of the present invention and are not intended to be limiting.
[0041]
(Embodiment 1)
In the present embodiment, a substrate having a photoresist coated on the surface is used as a gene sensor. As the photoresist, a positive photoresist that dissolves by light exposure or a negative photoresist that polymerizes by light exposure can be used.
[0042]
FIG. 1 shows an outline of the present embodiment using a positive photoresist and an outline of a gene sensor used therefor. As shown, the substrate 101 includes a support layer 106 and a positive photoresist layer 105. As the support layer 106, a semiconductor material typified by single crystal silicon, amorphous silicon, silicon carbide, silicon oxide, silicon nitride, etc .; inorganic materials such as glass, quartz glass, alumina, sapphire, forsterite, ceramics; and polyethylene, ethylene , Polypropylene, polyisobutylene, polyethylene terephthalate, unsaturated polyester, fluorine-containing resin, polyvinyl chloride, polyvinylidene chloride, polyvinyl acetate, polyvinyl alcohol, polyvinyl acetal, acrylic resin, polyacrylonitrile, polystyrene, acetal resin, polycarbonate, polyamide, Phenol resin, urea resin, epoxy resin, melamine resin, styrene / acrylonitrile copolymer, acrylonitrile / butadiene styrene copolymer, silica Over down resin, may be used an organic material such as polyphenylene oxide and polysulfone. Preferably, amorphous silicon or glass is used as the support layer 106.
[0043]
As the positive photoresist layer 105, a novolac resin-diazonaphthoquinone (DNQ) based resist based on a novolak resin, a copolymer of polymethyl methacrylate (PMMA) and PMMA, polymethylene sulfone, polyhexafluorobutyl methacrylate, Materials such as dissolution inhibitor-based resist = cholic acid-0-nitrobenzyl ester, such as polymethylisopropenyl ketone (PMIPK), radiolytic polymer resist = poly (1-trimethylsilylpropyne bromide) can be used. Preferably, a novolak resin-diazonaphthoquinone (DNQ) resist is used.
[0044]
In measurement, the nucleic acid probe 102 denatured into a single strand is immobilized on the support layer 106 of the substrate 101 at a predetermined interval by a method such as silane coupling. The immobilization of the nucleic acid probe can be performed using any method known to those skilled in the art. The term “gene sensor” used in this specification is used to refer to a substrate on which a nucleic acid probe is immobilized. In the upper left of FIG. 1, the analyte nucleic acid 103 is shown with a light shielding substance 104 bound as a labeling substance. The light-shielding substance 104 is selected from inorganic materials such as colored pigments such as titanium white, carbon black, zinc white, fumes, yellow lead, and toluidine blue, or iron, cobalt, zinc, titanium, tin, aluminum, nickel, and manganese. Mixtures of metals, and complexes of these metals can be used. The light blocking material 104 can be bound to the analyte nucleic acid 103 using any method known to those skilled in the art. Alternatively, as shown in the upper right of FIG. 1, when the analyte nucleic acid 103 and the single-stranded nucleic acid probe 102 are hybridized, they are specifically inserted only into the double strand having a light-shielding group that shields light. An intercalating agent 108 may be present and introduced into the formed duplex. An example of such an intercalating agent is a double-stranded intercalating agent having a flat intercalating group such as a phenyl group. Examples include a platinum complex such as a bipyridine platinum complex and a terpyridine platinum complex, a chromium complex, a zinc complex, and a cobalt complex. The conditions for hybridization between the nucleic acid probe and the analyte nucleic acid are known to those skilled in the art and are described in, for example, documents such as Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor (1982).
[0045]
After hybridization, the light source 109 irradiates the surface area of the substrate with ultraviolet light. The positive photoresist layer 105 is dissolved by exposure to ultraviolet light. On the other hand, a region where the light-shielding modifier 104 or the light-shielding insertion agent 108 exists, that is, a region on the substrate where the formed double strand is captured (hereinafter referred to as a double-stranded formation region) is shielded from light. Layer 105 remains undissolved as shown at the bottom of FIG.
[0046]
Here, the AFM driving device 112 scans the concavo-convex shape formed by the positive photoresist layer remaining on the surface of the support layer 106 with the AFM probe 113 or the like. The AFM (Atomic Force Microscope) is an apparatus devised by Dr. Vinich, who invented STM, in collaboration with Professor Kuwait of Stanford University in the United States to measure the surface shape of an insulator with STM. In the AFM, a small soft lever is inserted between the STM probe and the insulator sample surface. Here, a laser beam is incident on the back surface of the AFM probe 107 obliquely from the laser light source 109, the change in the reflection angle of the laser beam due to the probe displacement is read using the position analyzer 111, and the surface irregularity shape of the support layer 106 is obtained. By knowing, hybridization between the nucleic acid probe and the analyte nucleic acid can be detected.
[0047]
In addition, the board | substrate 101 in which such uneven | corrugated shape was engraved can preserve | save the shape semipermanently, for example by baking for 10 minutes at 120 degreeC, and as needed, as a test sample database, as needed Can be used.
[0048]
FIG. 2 shows an outline of a modified example of the present invention in which a positive resist is used as a mask and the support layer is further etched in addition to the embodiment shown in FIG.
[0049]
The configuration of the substrate is the same as the example shown in FIG. After hybridization, the mask is patterned using the positive photoresist 105 only in the double-strand formation region 213 formed by irradiating the surface of the substrate 101 with ultraviolet light from the light source 109. Thereafter, the support layer 106 is further processed by etching or the like, so that the uneven shape of the double-stranded formation region is enlarged in the support layer 106 as seen in the support layer shape 114 shown in the lower part of FIG.
[0050]
By further performing such an etching step, i) the uneven shape of the double-stranded formation region is enlarged, and the sensitivity of the shape measurement is increased, and ii) the test layer database is processed by processing the support layer As a result, it is possible to improve the storage stability of the substrate.
[0051]
Etching is performed by any method known to those skilled in the art, and either dry etching or wet etching can be applied thereto. As the etchant that corrodes the support layer, an isotropic etchant or an anisotropic etchant may be selected as necessary. As an isotropic wet etchant, for example, a silicon oxide film (SiO 2) obtained by thermally oxidizing single crystal silicon (Si) is used.₂) As the support layer 106, hydrofluoric acid (HF) and ammonium fluoride (NH_FourF) is known. For example, when polysilicon (p-Si) is used as the support layer 106 as an isotropic dry etchant, CF_FourGas is known. In the case where single crystal silicon is used as the support layer 106 as an anisotropic wet etchant, potassium hydroxide, hydrazine, EPW (eletindiamine-pyrocatechol-water), TMAH (tetramethylammonium hydroxide) and the like are known.
In either case, anisotropy is realized by utilizing the difference in etching rate with respect to the crystal plane, but TMAH is preferably used. CF as an anisotropic dry etchant_FourIt is known that anisotropic etching is realized by a mixed gas with other components mainly containing a gas.
[0052]
FIG. 3 shows a substrate support layer shape 115 obtained when the isotropic etching is performed and a substrate support layer shape 116 obtained when the anisotropic etching is performed. As shown in FIG. 3, in anisotropic etching, the support layer is processed along its crystal plane, so the double-stranded formation region has an enlarged convex shape (a plateau shape with a large bottom area). As expanded.
[0053]
Next, an example using a negative photoresist will be described.
[0054]
FIG. 4 is an outline of the method of the present invention using a negative photoresist, and an outline of a gene sensor used therefor. Compared with the case where the positive photoresist is used, the structure of the substrate is the same except that the photoresist layer is a negative photoresist layer 417. As a material for the negative photoresist layer 417, a cyclized polyisoprene-aromatic bisazide resist, a phenol resin-aromatic azide compound resist, polyvinylphenol-3, 3′-diazidodiphenylsulfone, polymethacrylic acid, which are UV resists, are used. Glycidyl and the like can be used. An azide compound having a cyclized polyisoprene as a base polymer, such as 2-6-di (4-didobezar) -4-methylcyclohexanone, is preferably used.
[0055]
In this example, after hybridization, the negative photoresist layer 417 is photopolymerized by irradiation with ultraviolet light from a light source 109 (not shown). On the other hand, since the region where the light shielding modifier 104 or the light shielding insertion agent 108 exists is shielded from light, the negative photoresist layer 417 is dissolved without being photopolymerized.
[0056]
Even when a negative photoresist layer is used, the shape of the double-stranded formation region can be expanded by further etching the support layer. This is achieved by etching the support layer using a negative resist as a mask. Only in the double-strand formation region, the mask is patterned using a negative photoresist 417. Thereafter, the support layer 106 is processed by etching, so that the depth of the recessed portion formed in the double-stranded formation region is expanded in the support layer 106 as seen in the support layer shape 418 shown in the lower part of FIG. Is done.
[0057]
By further performing such an etching step, even when using a negative photoresist, i) the uneven shape of the double-stranded formation region is enlarged, and the sensitivity of shape measurement is increased, and ii) support By processing the layer, an effect is obtained that the storability of the substrate can be enhanced as a database of test samples.
[0058]
FIG. 5 shows a shape 519 of the substrate support layer when the isotropic etching is performed and a shape 520 of the substrate support layer when the anisotropic etching is performed. As shown in FIG. 5, in the isotropic etching, the support layer is processed along its crystal plane, and the double-stranded region is enlarged as a concave shape.
[0059]
(Gene sensor production example and use)
Single crystal silicon was used for the support layer 106 of the substrate 101. First, the support layer 106 containing single crystal silicon was baked in a clean oven for 20 minutes. After dehydrating the surface of the support layer 106, using a spinner, a photoresist was spin-coated at 3500 rpm for 30 seconds, and baked at 90 ° C. for 10 minutes.
[0060]
The obtained substrate 101 was immersed in NaOH + 95% ethanol at room temperature for 2 hours with gentle shaking. Next, after rinsing three times with distilled water, the substrate 101 was immersed in a diluted solution of poly L-lysine (P8920, manufactured by Sigma) for 1 hour. The substrate 101 is taken out from this diluted solution, centrifuged at 500 rpm for 1 minute using a microtiter plate centrifuge, the remaining poly L-lysine diluted solution is removed, put into a suction thermostat, and dried at 40 ° C. for 5 minutes. I let you.
[0061]
On the surface of the obtained substrate 101, as a nucleic acid probe, 10 μM K-rasDNA ligand (5′-CCA-CCA-GCT-CCG-5 ′) / TE buffer solution (10 mM Tris, 1 mM EDTA (ethylenediaminetetraacetic acid)) The single-stranded DNA was immobilized on the substrate surface by spotting 10 μl of the solution and air drying. A, T, G, and C each represent a base sequence of a DNA nucleic acid.
[0062]
Next, after washing the substrate with pure water, 50 μl of 10 μM K-rasDNA ligand (5′-CCA-CCA-GCT-CCG-5 ′) / TE buffer solution was added as an analyte nucleic acid in a 65 ° C. constant temperature bath. The hybridization reaction was performed for 10 hours. Gently place the substrate in 0.03% SDS, SSC (sodium chloride, sodium citrate), gently wash, remove, and centrifuge at 500 rpm for 1 minute using a microtiter plate centrifuge to remove moisture. It was. It was then exposed for 4 seconds and developed for 70 seconds. The obtained substrate was rinsed with pure water for 30 seconds and then baked at 120 ° C. for 10 minutes. Using the AFM probe 707, a 2 μm × 2 μm region on the obtained substrate was scanned, and the surface shape was measured.
[0063]
(Embodiment 2)
In the present embodiment, a photocurable resin is used, and the photocurable resin is aggregated in the double-strand formation region of the gene sensor to form a convex portion on the substrate.
[0064]
FIG. 6 shows an outline of the present embodiment and a substrate used therefor. The substrate 601 includes a support layer 606. As a material used for the support layer 606, a semiconductor material typified by single crystal silicon, amorphous silicon, silicon carbide, silicon oxide, silicon nitride, or the like; an inorganic material such as glass, quartz glass, alumina, sapphire, forsterite, or ceramics; And polyethylene, ethylene, polypropylene, polyisobutylene, polyethylene terephthalate, unsaturated polyester, fluorine-containing resin, polyvinyl chloride, polyvinylidene chloride, polyvinyl acetate, polyvinyl alcohol, polyvinyl acetal, acrylic resin, polyacrylonitrile, polystyrene, acetal resin, Polycarbonate, polyamide, phenolic resin, urea resin, epoxy resin, melamine resin, styrene / acrylonitrile copolymer, acrylonitrile / butadienes Ren copolymer, silicone resin, organic materials such as polyphenylene oxide and polysulfone can be used.
Preferably, quartz glass is used as the substrate material.
[0065]
In the measurement, as in the first embodiment, the nucleic acid probe 102 denatured into a single strand is immobilized on the support layer 606 at a predetermined interval by silane coupling or the like. Here, the photo-curing resin 604 is bound to the analyte nucleic acid 103 in advance (shown on the upper left in FIG. 6). Alternatively, when the analyte nucleic acid 103 and the single-stranded nucleic acid probe 102 are hybridized, it is formed in the presence of an insertion agent 605 that has a functional group that causes photocuring and is specifically inserted only into the double strand. (Shown on the upper right in FIG. 6). In either case, a material having a photocurable property that cures when exposed to light will be present specifically only at the position where the double strand is formed. As the photocurable resin 604, one or more photopolymerization initiators selected from benzophenone or a substituted derivative thereof, benzoin or a substituted derivative thereof, acetophenone or a substituted derivative thereof, an oxime-based compound such as benzyloxime, and the like. Polyester acrylates, epoxy acrylates, urethane acrylates, and the like that are contained by weight can be suitably used. In addition, if necessary, the photo-curable resin 604 is appropriately formed of a chemical substance having a co-catalytic role such as a sensitizer, a filler, an inert organic polymer, a leveling agent, a thixotropic agent, and a thermal polymerization inhibitor. May be included.
[0066]
After the hybridization reaction, the light source 609 irradiates the substrate with light. Depending on the photocurable resin used, the substrate is irradiated with light having a wavelength of 200 nm to 800 nm. Ultraviolet light is preferably used. The resin cured by light irradiation aggregates in the double-stranded formation region on the substrate 601 to form a convex aggregate 613.
[0067]
Here, the shape of the aggregate formed on the surface of the support layer 601 is scanned by the AFM driving device 112 with the AFM probe 113 or the like. Here, the back surface 107 of the AFM probe is irradiated with laser from the laser light source 109, and the position of the laser light source 109 that follows the AFM probe 113 is read using the analyzer 111, whereby the probe and the analyte nucleic acid are high. Hybridization can be detected.
[0068]
Such a substrate having the aggregate 613 can also be stored semipermanently, for example, by baking at 120 ° C. for 10 minutes, and can be used as a test sample database as needed.
[0069]
(Production example and use of gene sensor)
As the support layer 606, a slide glass (3010 slide glass manufactured by Gold Seal Brand) was used. The slide glass was immersed in a NaOH + 95% ethanol solution at room temperature for 2 hours, and then rinsed three times with distilled water. Next, the slide glass was immersed in a diluted solution of poly L-lysine (manufactured by P8920 Sigma) for 1 hour. The slide glass was taken out from the poly L-lysine dilution, centrifuged at 500 rpm for 1 minute in a microtiter plate centrifuge to remove the excess poly L-lysine dilution, and then placed in a suction-type thermostat, at 5 ° C. for 5 minutes. Let dry for minutes.
[0070]
UV curable resin EHA (2-ethylhexyl acrylate, CH₂= CHCOOCH₂CH (C₂H_Five) C_FourH₉) At the end of the acryloyl group (CH₂= CHCO-) and 10 μM 5'-aminated K-ras DNA (5'-H₂N-C₆H₁₂-CGG-AGC-TGG-TGG-3 ') (A, T, G, C indicate the base sequence of DNA) was used for labeling. Photopolymerization monomer (EHA) -labeled K-rasDNA / TE buffer solution (10 mM Tris, 1 mM EDTA (ethylenediaminetetraacetic acid)) is spotted on a dried glass slide substrate to immobilize single-stranded DNA on the substrate surface. (Gene sensor). Furthermore, after washing this gene sensor with pure water, 50 μl of 10 μM K-rasDNAligand (5′-CCA-CCA-GCT-CCG-3 ′) / TE buffer solution was added and soaked for 10 hours to perform hybridization. Double-stranded DNA was formed on the surface.
[0071]
Subsequently, the gene sensor was exposed to ultraviolet rays (exposure time: 4 seconds × twice). And after developing for 70 seconds, it rinsed for 30 seconds with the pure water. After baking the washed gene sensor at 120 ° C. for 10 minutes. Using the AFM probe and the AFM driving device, the shape of the aggregate formed on the surface of the support layer was scanned.
[0072]
In the first and second embodiments, as the photoresist, cyclized rubber that is a photoresist for light exposure, polycinnamic acid, novolac resin, or the like as a main material, or a ring that is a photoresist for far ultraviolet. Made mainly of synthetic rubber, phenolic resin, polymethylisopropenyl ketone (ΡMΙPK) and polymethylmethacrylate (ΡMM な ど), etc., made mainly of CO ア ク リ レ ー ト and metal acrylate, which are X-ray photoresists, or thin film Listed in the above handbook, including ΡMMΡ, which is an electron beam resist, as described in the Handbook (Japan Society for the Promotion of Science Thin Film 131st Edition, 1st edition, December 10, 1983, Ohmsha) Any photoresist can be selected and used according to the purpose.
[0073]
(Embodiment 3)
In this embodiment, an AFM probe (AMF cantilever) is used, and an electric charge is applied to this and tapped to dent the double-chain formation region of the plastic deformation layer disposed on the substrate to form a recess. , Measure its shape change.
[0074]
FIG. 7 shows an outline of a device used in this embodiment. As shown in FIG. 7, the base of the AFM probe 706 is joined to the piezoelectric element 707, and the AFM probe 706 can be vibrated by applying a voltage from the power source 708 to the piezoelectric element 707. The voltage to be applied is usually a voltage in the range of 0.1 mV to 100 V. The amplitude with which the AFM probe 706 vibrates is usually in the range of 10 nm to 100 μm. As the piezoelectric element 707, any single layer type, bimorph type, or multilayer type piezoelectric element known to those skilled in the art can be used, and in particular, a bimorph type piezoelectric element is preferably used. The substrate 701 includes a support layer 704 and a plastic deformation layer 705.
[0075]
As the support layer 704, a semiconductor material typified by single crystal silicon, amorphous silicon, silicon carbide, silicon oxide, silicon nitride, or the like; and an inorganic material such as glass, quartz glass, alumina, sapphire, forsterite, or ceramics can be used. .
[0076]
As the plastic deformation layer 705, polyethylene, ethylene, polypropylene, polyisobutylene, polyethylene terephthalate, unsaturated polyester, fluorine-containing resin, polyvinyl chloride, polyvinylidene chloride, polyvinyl acetate, polyvinyl alcohol, polyvinyl acetal, acrylic resin, polyacrylonitrile, Suitable organic materials such as polystyrene, acetal resin, polycarbonate, polyamide, phenol resin, urea resin, epoxy resin, melamine resin, styrene / acrylonitrile copolymer, acrylonitrile / butadiene styrene copolymer, silicone resin, polyphenylene oxide and polysulfone Used. More preferably, a polycarbonate or a polycarbonate coated with a PMMA resin is used as the plastic deformation layer 705.
[0077]
A nucleic acid probe is immobilized on the plastic deformation layer 705. After hybridization, the substrate is dried and the AFM probe 706 is in soft contact with the sample surface. The contact pressure is adjusted in the range of 0.1 nN to 1 mN, but a contact pressure of about 100 nN is preferably used. If necessary, the back surface of the AFM probe is irradiated with laser light at the double-strand formation position, and the probe is heated. Alternatively, the laser light applied by the laser light source 709 can directly irradiate the tip surface of the AFM probe 706. As laser, solid-state semiconductor laser, CO₂A laser, a YAG laser, an excimer laser, or the like can be used. An excimer laser having a wavelength of around 200 nm can be suitably used.
[0078]
The heat of the heated probe is transmitted to the surface of the gene sensor, and is used as energy for dissociating the double strand in the double strand formation region. Therefore, compared to the region other than the double strand formation region, Due to an increase in the plastic deformation yield point, the concave structure (nanometer size) formed by heating in the double-stranded formation region is formed by heating in the region where the single-stranded probe nucleic acid is immobilized. It becomes smaller than the concave structure of the substrate surface. The outline is shown in FIG. By reading the concavo-convex structure on the surface of the substrate thus formed, the double-stranded formation reaction can be detected. When reading such a concavo-convex structure on the surface of the substrate, the back surface of the AFM probe 706 is irradiated with laser light having a low output from a laser light source 709, and the reflected laser light is amplified by a photomultiplier 711 to detect the position. To the detector 712 for detection (optical lever detection method). Since the plastic deformation yield point of the plastic deformable material at normal temperature is large, the substrate surface does not undergo plastic deformation.
[0079]
(Examples of gene sensor production and use)
As the support layer 704, a single crystal silicon piece having a size of 30 mm square and a thickness of 525 μm was used. This single crystal silicon piece is thermally oxidized at 600 ° C.₂A layer was formed to obtain a sensor substrate. Thereafter, PMMA (methyl methacrylate) resin was deposited to a thickness of 2 μm. K-rasDNAligand (5'-CCA-CCA-GCT-CCG-5 ') was dissolved in 100 µl of TE buffer solution (10 mM Tris, 1 mM EDTA (ethylenediaminetetraacetic acid)) to a final concentration of 10 µM. Next, aminopropyltriethoxysilane was used to activate the side chain of the PMMA resin on the substrate, and K-rasDNAligand was cross-linked at two sites.
A, T, G, and C represent DNA base sequences.
[0080]
After the DNAligand-crosslinked substrate was washed with pure water, 50 μl of 10 μM K-rasDNAligand (5′-CCA-CCA-GCT-CCG-5 ′) / TE buffer solution was added to one of the two locations, The substrate was immersed for 10 hours and hybridized to form double-stranded DNA on the substrate surface.
[0081]
An AFM probe is brought into contact with this substrate at a pressure of 100 nN, the back surface of the AFM probe is irradiated with a semiconductor laser beam having a spot diameter of 1 μm, and 50 V is applied to the piezoelectric element to vibrate the AFM probe. A region of 2 μm × 2 μm was scanned for each of a total of three places on the strand formation region, the single-stranded K-rasDNAligand immobilization region, and the PMMA resin surface. Thereafter, the laser irradiation was stopped, scanning was performed at a contact pressure of 10 nN, and the surface unevenness shape in the range of 2 μm × 2 μm at the same three locations on the substrate was measured.
[0082]
As a result of the measurement, the diameter of the opening of the recess formed in the double-stranded formation region was 100 nm. On the other hand, the diameter of the opening of the concave portion of the single-stranded K-rasDNAligand fixing region was slightly small. Since the contact pressure of the AFM probe is constant, the recess depth is proportional to the diameter of the opening of the recess. Since the diameter of the opening of the recess was 100 nm, if the gene was immobilized on the substrate at a fixed pitch of 200 nm, 1.5 × 10⁸Sample / cm²It is possible to detect genes at high density.
[0083]
(Embodiment 4)
In this embodiment, an AFM probe (AFM cantilever) having a conductive layer is used, a charge is applied to this, and the double strand formation region of the gene sensor is scratched and thermally expanded to form a convex structure.
[0084]
FIG. 9 shows an outline of this embodiment and a gene sensor. As shown in FIG. 9, the AFM cantilever 907 includes a conductive layer 908, and thus the probe has a conductive property. For example, the conductive layer 908 can be formed by forming a metal film on the base of the AFM cantilever 907. As a film forming method, vapor deposition, sputtering, CVD, or the like can be used, and a vacuum vapor deposition method is preferably used. Alternatively, the semiconductor probe may be doped with boron or the like to have conductivity.
[0085]
The substrate 901 includes a support layer 904, a conductive layer 905, and a plastic deformation layer 906. As described above, the support layer 904 includes a semiconductor material typified by single crystal silicon, amorphous silicon, silicon carbide, silicon oxide, silicon nitride, and the like; and inorganic materials such as glass, quartz glass, alumina, sapphire, forsterite, and ceramics. It can be a material.
[0086]
As the conductive layer 905, an electrode containing a noble metal such as platinum, platinum black, gold, palladium, rhodium, silver, mercury, or tungsten is preferably used, and graphite, glassy carbon, pyrolytic graphite, carbon paste, carbon, A carbon electrode typified by —bon fiber—and oxide electrodes such as titanium oxide, tin oxide, and manganese oxide can be used.
[0087]
Plastic deformation layer 906 includes polyethylene, ethylene, polypropylene, polyisobutylene, polyethylene terephthalate, unsaturated polyester, fluorine-containing resin, polyvinyl chloride, polyvinylidene chloride, polyvinyl acetate, polyvinyl alcohol, polyvinyl acetal, acrylic resin, polyacrylonitrile. Organic materials such as polystyrene, acetal resin, polycarbonate, polyamide, phenol resin, urea resin, epoxy resin, melamine resin, styrene / acrylonitrile copolymer, acrylonitrile / butadiene styrene copolymer, silicone resin, polyphenylene oxide and polysulfone are suitable. Used for. More preferably, polystyrene is used as the plastic deformation layer 906.
[0088]
The nucleic acid probe 102 is immobilized on the plastic deformation layer 906 in the same manner as described above. After hybridization, scanning is performed while the AFM probe 907 is in soft contact with the sample surface. The contact pressure is adjusted in the range of 0.1 nN to 1 mN, but a contact pressure of about 60 nN is preferable. By making the AFM probe contact and scan, the plastic deformation layer is scratched. At that time, a high frequency voltage is applied between the conductive layer 905 of the substrate 901 and the conductive layer 908 of the AFM probe 907 by the voltage application device 913, so that the polymer in the plastic deformation layer is plasticized by heat near the probe. Raises by deforming. This voltage is applied by the voltage application device 913 between the conductive layer 905 of the substrate 901 and the conductive layer 908 of the AFM probe 907. Usually, a voltage of 1 KHz to 100 MHz in the range of 0.1 mV to 100 V is applied. Preferably, a high frequency voltage around 50 MHz is applied.
[0089]
In general, the ridge height depends on the amount of dielectric loss of the polymer. Since the nucleic acid itself is a non-conductor, the dielectric constant between the probe and the substrate is higher in the double-stranded formation region than in the single-stranded nucleic acid probe fixing region, and the amount of dielectric loss is increased. That is, the convex structure (nanometer size) of the sample surface formed in the double-stranded formation region is larger and higher than the convex structure formed in the single-stranded nucleic acid probe immobilization region.
[0090]
The concavo-convex structure formed on the surface of the substrate thus formed can be read to detect a double strand formation reaction.
[0091]
When reading the uneven structure on the substrate surface, as shown in FIG. 10, the back surface of the AFM probe 907 is irradiated with a laser beam 909 having a weak output from the laser light source 909, and the signal is amplified by the photomultiplier 911. This is transmitted to the position detector 912. As described above, the reading is preferably performed by the optical lever detection method.
[0092]
(Production example and use of gene sensor)
Quartz glass was used as the support layer 904 of the substrate 901. Silver was deposited on the support layer 904 by vacuum deposition to form a conductive layer 905. Next, a benzene solution of polystyrene was spin-coated thereon and annealed at 95 ° C. for 20 hours to produce a plastic deformation layer 906. Similar to Embodiments 1 and 2 (manufacturing example and use of gene sensor), after conducting nucleic acid probe immobilization and hybridization reaction, a conductive layer containing silver doped with boron and made conductive is provided. The AFM probe was contacted at a contact pressure of 60 nN, and a 2 μm × 2 μm region was scanned while being applied at 50 MHz. When the applied voltage was 8 V, a protrusion of about 10 μm was observed.
[0093]
(Embodiment 5)
FIG. 11 is a diagram showing an outline of the automatic gene detection apparatus of the present invention. As shown in FIG. 11, the measuring apparatus for gene detection of the present invention includes a gene sensor 33 to which a nucleic acid probe is immobilized, means 43 for moving the gene sensor, and a sample containing a gene nucleic acid sample denatured into a single strand. A reservoir 31 for storing the solution, a reaction tank 32 for performing hybridization between the gene nucleic acid sample and the nucleic acid probe on the gene sensor, a temperature control device 34 for controlling the temperature of the sample solution, and after the hybridization reaction, the gene Cleaning means 29 for cleaning the sensor to remove unreacted sample solution, means 38 (exposure device, voltage application device, etc.) for applying energy to a region on the gene sensor 33, and the geometric shape of the gene sensor A device 37 for measuring changes is provided. The energy applying means 38 can be controlled by the control device 39 according to a signal generated by the computer 41, for example. Reference numeral 36 is a cantilever for scanning the gene sensor 33 arranged as necessary.
[0094]
Furthermore, the automatic gene detection apparatus of the present invention stores a solution containing a labeling substance, and two bottles formed during the hybridization reaction between the labeling substance, the nucleic acid probe and the gene nucleic acid sample, or on the surface of the gene sensor. A labeling substance reservoir 42 may be provided for storing the labeling substance so as to bind to the double-stranded nucleic acid by reacting with the strand nucleic acid. The labeling substance reservoir 42 is a member schematically indicated by reference numeral 30 in FIG. 11, which is a nucleic acid dissociation device that is installed as necessary, for separating the double strand formed on the gene sensor. This is a member for enabling reuse of the gene sensor. Reference numeral 35 is a moving stage for moving the gene sensor, and the gene sensor is moved to the washing means 29, the sample solution reservoir 31, the reaction tank 32, and the nucleic acid dissociation apparatus 30, respectively.
[0095]
As the device 37 for measuring the geometric shape change of the gene sensor, for example, a stylus type surface roughness meter can be adopted. In this stylus type surface roughness meter, the probe 36 is composed of a tuning fork type crystal resonator, a microfork for electronic circuits, a piezoelectric material represented by PZT, or a ceramic having piezoelectricity. Alternatively, a tapping stylus (vibrating stylus type surface roughness meter) having a vibrating mechanism may be used.
[0096]
Alternatively, an optical surface shape measuring device, particularly a scanning probe microscope (SPM) is preferably used as the device 37 for measuring the geometrical shape change of the gene sensor.
[0097]
As used herein, the term “scanning probe microscope” is a generic term for scanning microscopes capable of observation at the atomic level. More specifically, a scanning tunneling microscope (STM), an atomic force microscope (AFM), a friction force microscope, a Maxwell stress microscope, a magnetic force microscope, a photon scanning tunneling microscope, a scanning proximity microscope Examples thereof include a near-field optical microscope (SNOM), a scanning near-field acoustic microscope, and the like. AFM is classified into repulsive type, attractive type and tapping type ("Tapping type" is a registered trademark of Digital Instruments, Inc., located in Santa Barbara, California, USA). New microscopes such as force microscopes, Maxwell stress microscopes, magnetic force microscopes, photon scanning tunneling microscopes, photon scanning tunneling microscopes, and scanning near-field optical microscopes (SNOMs) have been developed. Any scanning probe microscope can be applied to the automatic gene detection apparatus of the present invention.
[0098]
Techniques relating to STM are described in, for example, literature (G. Binning et al., IBM J. RES. DEVELOP., 30 (4): 355-369, 1986; Conrad Schneiker et al., J. of Microscopy, 152 (2): 585-596, 1988; H. Kaisuka, Rev. Sci. Insrum., 60 (10): 3119-3122, 1989).
[0099]
In addition, a technique related to AFM is described in the literature (G. Binnig et al., Physical Review Letters, 56 (9): 930-933, 1986; TR Albrecht et al., J. Appl. Phys., 62 (7): 2599-). 2602, 1987; AL Weisenhorn et al., Appl. Phys. Lett., 54 (26): 2651-2653, 1989; T. R. Alblecht et al., J. Vac. Sci. Technol., A8 (4): 3386-3396, 1990; RC Barrett et al., Rev. Sci.Instrument., 62 (6), 1991).
[0100]
Techniques relating to SNOM are described, for example, in the literature (Pohl et al., J. of Microscopy, 152 (3): 853-861, 1988; Constant AJ Putman et al., Appl. Phys. Lett., 64 (18): 2454-. 2456, 1994; NF van Hulst, Appl. Phys. Lett., 62 (5): 461-463, 1993).
[0101]
Reference techniques for measuring and imaging biological materials using these SPM techniques are described in the literature (Zasadzinski et al., Science 239: 1013-1015, 1988; Emch et al., J. of Microscopy, 152 ( 1): 85-92, 1988; Marti et al., J. of Microscopy, 152 (3): 803-809, 1988; Drake et al., Science, 243: 1586-1589, 1989; Gould et al., J. Vac. Technol., 8 (1): 369-373, 1990; Jericho et al., J.Vac.Sci.Technol., 8 (1): 661-666, 1990; Coragtger et al., Micron, 25 (4): 371-385. 1994; You , Micron, 26 (4): 311-315,1995) have been described in.
[0102]
Furthermore, reference techniques for measuring nucleic acid molecules by SPM techniques are described in the literature (Feng et al., J. of Microscope, 152 (3): 811-816, 1988; Driscoll et al., Nature, 346: 294-296, 1990. Fairtel and Beveridge, Micron, 26 (4), 347-362, 1995; Yang and Shao, Micron, 26 (1): 35-49, 1995; Rivetti et al., J. Mol. Biol., 264: 919-932. 1996).
[0103]
The detailed operation technique of the SPM applied to the method of the present invention can be referred to as appropriate from the above documents. The scanning probe microscope used in the automatic gene detection apparatus of the present invention is composed of a means for applying a voltage between a probe or a probe and a gene sensor substrate, or a piezoelectric material represented by PZT or a ceramic having piezoelectricity. And a mechanism for vibrating the probe. Alternatively, a means for irradiating either the front surface or the back surface of the gene sensor near the tip of the probe may be provided.
[0104]
Typically, in the automatic gene detection device of the present invention, the cantilever 36 is scanned using the gene sensor moving means 43 and the geometric shape of an arbitrary region of the gene sensor 33 can be measured. This can be driven by a movement control device 40 and a computer 41 that control the gene sensor movement device 43. The computer 41 includes a program for controlling scanning of the cantilever 36, a program for arbitrarily controlling the light quantity and voltage value of the energy applying means 38, a program for acquiring, synthesizing and processing a surface image, a double-stranded nucleic acid hybridization amount or A program for digitizing, visualizing, calculating, and statistically processing the amount of dissociation and the like, and a program for automating these measurements may be provided.
[0105]
The details of the above-described constituent members of the automatic gene detection device of the present invention and the connection thereof are not particularly described in detail, but can be configured according to the knowledge of a person skilled in the art by employing known techniques in the field.
[0106]
Although one embodiment of the copper clad laminate according to the present invention has been described, the present invention is not limited to this at all, and various modifications can be made based on the knowledge of those skilled in the art without departing from the spirit of the present invention. The present invention can be implemented in the form of improvements, changes and modifications. Although the present invention has been described with DNA as a representative example, the present invention is also applicable to the processing and analysis of RNA, proteins, polysaccharides, lipids and other macromolecules. The references cited in this specification are hereby incorporated by reference in their entirety, including the nucleic acid and amino acid sequences listed in each reference, and are hereby expressly incorporated by reference. It is considered.
[0107]
【The invention's effect】
  Ability to accumulate analytes in solution at high density and detect them easily and with high sensitivityLawYoDressA device is provided.
[Brief description of the drawings]
FIG. 1 is a diagram showing an outline of Embodiment 1 of the present invention using a positive photoresist.
FIG. 2 is a diagram showing an outline of a modification of the first embodiment of the present invention using a positive photoresist.
FIG. 3 is a diagram showing a shape-change gene sensor in a modified example of Embodiment 1 of the present invention using a positive photoresist.
FIG. 4 is a diagram showing an outline of Embodiment 1 of the present invention using a negative photoresist.
FIG. 5 is a diagram showing a shape-changed gene sensor according to a modification of the first embodiment of the present invention using a negative photoresist.
FIG. 6 is a diagram showing an outline of Embodiment 2 of the present invention using a photocurable resin.
FIG. 7 is a diagram showing an outline of Embodiment 3 of the present invention using a plastically deformed layer.
FIG. 8 is a diagram schematically showing Embodiment 3 of the present invention using a plastically deformed layer.
FIG. 9 is a diagram schematically showing Embodiment 4 of the present invention using a layer that changes inelasticity.
FIG. 10 is a diagram schematically showing Embodiment 4 of the present invention using a layer that changes inelasticity.
FIG. 11 is a diagram showing an outline of an automatic gene detection apparatus of the present invention.
[Explanation of symbols]
101, 601, 701, 901 substrate
102 Nucleic acid probe (single strand)
103 Gene Nucleic Acid Sample
104 Labeling substance (shading substance)
105 Photoresist layer
106, 606, 704, 904 Support layer
108 Labeling substance (light-shielding insertion agent)
109 Light source
110 Light source driving device
111 Position detector
112 AFM drive
113,706 AFM probe
213 Double strand forming region
114 Support layer shape
115 Support layer shape
116 Support layer shape
417 negative photoresist
418 Support layer shape
519 Support layer shape
520 Support layer shape
604 photocurable resin
605 Insertion agent having a functional group that causes photocuring
613 Aggregate
705 Plastic deformation layer
707 Piezoelectric element
708 power supply
709, 909 Laser light source
710, 910 Laser drive device
711, 911 Photomaru
712, 912 Position detector
905 conductive layer
906 Plastic deformation layer
907 cantilever
908 conductive layer
913 Voltage application device
29 Cleaning means
30 Nucleic acid dissociation equipment
31 Sample solution reservoir
32 reactor
33 Gene sensor
34 Temperature controller
35 Moving stage
36 Cantilever
37 Substrate surface geometry measuring device
38 Exposure device / Voltage application device
39 Control device
40 Movement control device
41 computer
42 Labeled substance reservoir
43 Gene sensor moving means

Claims (7)

Capturing a target analyte in the nucleic acid probe on a substrate having a support layer, a photoresist layer disposed on the support layer, and a nucleic acid probe fixed to the support layer on the surface;
Applying energy selected from the group consisting of visible light, ultraviolet light, infrared light, X-rays, and electron beams to the surface of the substrate, modifying the photoresist layer in the region where the analyte is captured, An analyte measurement method comprising: forming an uneven shape on the substrate; and detecting the uneven shape with a scanning probe microscope, thereby detecting the presence of the analyte.   The analyte measuring method according to claim 1, wherein the step of forming a concavo-convex shape on the substrate is performed in the presence of a labeling substance that specifically binds to the analyte captured on the substrate.   The analyte measurement method according to claim 1, wherein the analyte has a labeling substance.   The analyte measurement method according to claim 1, wherein the analyte has a labeling substance made of a light shielding substance.   2. The analyte measuring method according to claim 1, wherein the analyte is a nucleic acid.   The analyte measurement method according to claim 1, further comprising a step of fixing the uneven shape of the substrate.   The analyte measurement method according to claim 1, further comprising a step of etching the support layer using the photoresist layer as a mask after the step of forming an uneven shape on the substrate.

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