WO2005033702A1 - Puce d'inspection, procede de production associe et procede d'inspection au moyen de ladite puce - Google Patents

Puce d'inspection, procede de production associe et procede d'inspection au moyen de ladite puce Download PDF

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Publication number
WO2005033702A1
WO2005033702A1 PCT/JP2004/014408 JP2004014408W WO2005033702A1 WO 2005033702 A1 WO2005033702 A1 WO 2005033702A1 JP 2004014408 W JP2004014408 W JP 2004014408W WO 2005033702 A1 WO2005033702 A1 WO 2005033702A1
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Prior art keywords
test
nucleic acid
chip
structural
inspection
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PCT/JP2004/014408
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English (en)
Japanese (ja)
Inventor
Kenji Yamaguchi
Mineo Sugiyama
Takatoshi Kinoshita
Yo Kikuchi
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Pokka Corporation
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Publication of WO2005033702A1 publication Critical patent/WO2005033702A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0697—Artificial constructs associating cells of different lineages, e.g. tissue equivalents
    • C12N5/0698—Skin equivalents
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2502/00—Coculture with; Conditioned medium produced by
    • C12N2502/09—Coculture with; Conditioned medium produced by epidermal cells, skin cells, oral mucosa cells
    • C12N2502/094—Coculture with; Conditioned medium produced by epidermal cells, skin cells, oral mucosa cells keratinocytes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2502/00—Coculture with; Conditioned medium produced by
    • C12N2502/13—Coculture with; Conditioned medium produced by connective tissue cells; generic mesenchyme cells, e.g. so-called "embryonic fibroblasts"
    • C12N2502/1323—Adult fibroblasts

Definitions

  • the present invention relates to a test chip used for testing a trace substance contained in a test mixture, a method for producing the same, and a test method using the chip.
  • a hybridization probe disclosed in Patent Document 1 As a test chip of this type, for example, a hybridization probe disclosed in Patent Document 1 is known.
  • the hybridization probe has a rod and a nucleic acid that binds to the rod and specifically binds to a target nucleic acid.
  • the nucleic acid RNA or single-stranded DNA having a base sequence complementary to the target nucleic acid is used.
  • This hybridization probe exhibits structural coloring based on the theory of multi-layer thin-film interference due to a rod-like body oriented in the form of a film. Then, when the specific hybridization between the RNA or the single-stranded DNA and the target nucleic acid is caused, the structural coloring of the hybridization probe is changed, and the specific probes and hybrids are changed. Formation is detected. Therefore, according to this probe, it is possible to directly and highly accurately and quantitatively evaluate the formation of special nuclei and hybrids in an aqueous or gaseous phase in a short time without requiring special techniques, and
  • Patent Document 2 discloses a structural variable body that causes a structural change by stimulation, a rod-shaped body having a length of 81 Onm or less, and a capturing structure that binds to the rod-shaped body and specifically captures a capturing target.
  • a capture body having a body is disclosed. This capturer exhibits structural color development as in the case of the hybridization probe.
  • an inclusion compound, an antibody, a nucleic acid, a hormone receptor, a lectin, or a physiologically active substance receptor is used as the capture structure.
  • the hybridization probe of Patent Document 1 has the advantage that a nucleic acid having a desired base sequence can be arbitrarily prepared and used, but the primary structure of the nucleic acid, that is, the sequence It was limited to the use of detecting information-only hybridization.
  • the primary structure it is possible to recognize three-dimensional tertiary structures in addition to the hybridization depending on the three-dimensional structure.
  • such a capture body has a drawback that it is difficult to artificially produce a desired capture structure like the nucleic acid in the hybridization probe. In particular, it has been extremely difficult to artificially create a capture structure that recognizes the three-dimensional structure of the capture target.
  • Patent Document 1 Japanese Patent Application Laid-Open No. 2002-345494
  • Patent Document 2 JP 2002-273219 A
  • a test chip used for testing a trace substance contained in a test mixture comprising: a structural coloring substrate; and a nucleic acid ligand bound to the structural coloring substrate.
  • the nucleic acid ligand is constituted by a nucleic acid having a higher-order structure.
  • a film forming step of forming a monomolecular film of a structural color body having a protective group on the surface of a supporting substrate, and removing the protective group from the formed monomolecular film comprising a charging step of charging the monomolecular film and a binding step of bonding a nucleic acid ligand to the charged monomolecular film.
  • FIG. 1 (a)-(d) is a schematic view showing a procedure for manufacturing a test chip and a test method according to the embodiment.
  • FIG. 2] (a)-(d) is a diagram showing an AFM topography image of the surface of the test chip of the example corresponding to the state of FIG. 1 (a)-(d), respectively.
  • FIG. 3 is a diagram schematically showing a reaction at the time of producing an abtamer chip of an example.
  • FIG. 4 is a graph showing the test results of the example.
  • the test chip 11 of the present embodiment includes a structural coloring substrate 12 and a nucleic acid ligand 13 bound on the structural coloring substrate 12.
  • the test chip 11 can easily, quickly and accurately detect a target 14 contained in a sample solution containing a test mixture (so-called specimen). In particular, it is possible to easily, quickly and accurately test the target 14 contained in a trace amount in the test mixture.
  • target molecules such as proteins, peptides, saccharides (polysaccharides), glycoproteins, lipids, carbohydrates, allergens, drugs, pesticides, and environmental hormones, or those target molecules are provided on the surface. And cells of multicellular organisms (including human and non-human organisms), unicellular organisms, and viruses.
  • the target molecule and the target 14 may be natural or artificial. Further, the target molecule may be any of an organic substance and an inorganic substance. These target molecules are particularly preferably biological constituent molecules or bioactive molecules, which are preferably organic substances, in order to easily carry out a specific and highly accurate test.
  • these target molecules form a specific three-dimensional structure such as a higher-order structure in order to more easily perform a specific and high-precision test.
  • a higher-order structure examples include a secondary structure, a tertiary structure, and a quaternary structure.
  • Examples of the test mixture containing such a target 14 include a sample for a food inspection test, a microorganism test, an environmental test, a toxicity test, or a component analysis test.
  • Examples of the sample include beverages such as milk-containing beverages and fruit juice-containing beverages, foods, cosmetics, quasi-drugs or pharmaceuticals, or raw materials thereof.
  • the samples include blood, urine, and saliva. Fluids, body fluids such as lymph fluid, specimens such as stool and biopsy tissue, and collections for environmental and toxicity tests can also be used.
  • the sample liquid containing the test mixture when the test mixture is a liquid such as a solution, a suspension, or an emulsion, it can be used as it is. In addition, it may be diluted with a solvent such as water or a buffer, or may be concentrated. When the test mixture is in a paste or solid form, it can be used by dissolving, suspending or emulsifying in a solvent such as water or a buffer.
  • the sample solution is diluted, concentrated, desalted, dialyzed, and pH-adjusted in order to maintain the three-dimensional structure of the target 14 and the nucleic acid ligand 13 (eg, salt concentration, pH, temperature). The following processing may be performed. At this time, the nucleic acid ligand 13 and the target 14 easily bind specifically on the test chip 11, so that the test accuracy is easily improved.
  • the conditions include physiological conditions or conditions milder than those conditions.
  • the physiological conditions include conditions under which microorganisms such as Escherichia coli, mold, Bacillus, staphylococci, yeast, and lactic acid bacteria, preferably single-cell organisms, can grow.
  • the mild condition refers to a condition that does not easily affect the three-dimensional structure of the target 14.
  • Such conditions include, for example, setting the pH of the sample solution within the range of 6-8, and setting the salt concentration to be equal to or lower than physiological saline.
  • the temperature of the sample solution is preferably higher than 0 ° C and not higher than 50 ° C, more preferably 10 to 40 ° C, still more preferably 20 to 37 ° C. .
  • the nucleic acid ligand 13 is composed of a nucleic acid having a higher-order structure such as a secondary structure or a tertiary structure, and specifically binds to the target molecule by the higher-order structure.
  • This nucleic acid ligand 13 is composed of single-stranded DNA or single-stranded RNA.
  • the term “specifically binds” means a bond (for example, biological bond) composed of a combination of various bonds such as a hydrogen bond, an intermolecular force (Van der Waals force) bond, a coordinate bond, and an ionic bond. I do. Therefore, the term “specific binding” means a reversible binding that does not involve the transfer of electrons, and excludes a shared binding.
  • the higher-order structure of the nucleic acid ligand 13 is formed by base sequence-dependent intramolecular hybridization such as a stem-loop structure. That is, this higher-order structure is A plurality of oligonucleotide sequences or oligonucleoside sequences dispersed in a molecule are formed by hybridizing with each other. Since the nucleic acid ligand 13 has a higher-order structure, it is not easily decomposed by DNase and RNase. The nucleic acid ligand 13 has a negative charge. As long as the nucleic acid ligand 13 forms a higher-order structure, it is possible to use a natural base sequence or an artificial base sequence with a deviation of! /.
  • an aptamer is most preferably used.
  • This aptamer is also composed of single-stranded DNA or single-stranded RNA of about 60 bases.
  • RNA abtamer composed of single-stranded RNA as shown in Fig. 3 It is most preferably used because it easily forms a higher-order structure.
  • a substance that binds to a bucket can be obtained by purifying a substance that binds to a bucket by the method of bystematic evolution of ligands (exponential enrichment).
  • a molecule that binds to a target is selected from a nucleic acid population (combinatorial library) having a random base sequence, and then the selected molecule is amplified by the polymerase chain reaction (PCR). In this method, selective amplification is repeated several rounds.
  • PCR polymerase chain reaction
  • the structural coloring substrate 12 includes a supporting substrate 21 and a large number of structural coloring members 23 arranged so as to form one layer on the surface of the supporting substrate 21. Indicates color development.
  • the structural coloring refers to coloring by a coloring mechanism represented by the structural color of the body surface of a morpho butterfly, a beetle, or a tropical fish.
  • This structural coloration is different from pigmentary coloration based on a chemical structure in which electrons are transferred and colored when irradiated with light, such as a dye or pigment, and the surface of an object has a wavelength equal to or less than a specific light wavelength.
  • This is a phenomenon in which a color is formed by having a fine structure.
  • This structural coloring is based on the multilayer thin-film interference theory, which is the basic principle of coloring of morpho butterfly wing scales, and involves at least one of light interference, diffraction and scattering power.
  • the thickness of the thin film and the refractive index thereof are changed. And to As a result, light of a specific wavelength is reflected accordingly, so that its color tone can be arbitrarily controlled like the skin of a chameleon.
  • the reflection of light of a specific wavelength is changed by bonding the target 14 to the surface of the thin film without substantially changing the external stimulus, that is, the electric field, magnetic field, temperature and light.
  • the binding state can be detected by visual or visible reflection analysis when using visible light, and can be detected by analyzing infrared or ultraviolet reflection spectrum when using infrared or ultraviolet light. It is.
  • the supporting substrate 21 is formed in a flat plate shape with a flat surface.
  • the support substrate 21 is not particularly limited as long as it is a material capable of fixing the structural color body 23 on the surface.
  • the support substrate 21 is preferably made of an organic material such as plastic or an inorganic material such as glass or quartz, because it is easy to mold. Further, since the structural color body 23 is easily fixed to a thin film exhibiting good structural color development, it is particularly preferable that the support substrate 21 be made of silicon or silica containing no impurities. .
  • the structural color body 23 shows structural color when immobilized on the surface of the support substrate 21 in a thin film form, and binds to both the support substrate 21 and the nucleic acid ligand 13.
  • a rod-shaped molecule is preferably used because structural color is easily exhibited. It is particularly preferable that the structural color body 23 has a rod shape having an aspect ratio of more than 10, since a rod shape having an aspect ratio of more than 1 is preferable since structural color development is more likely to occur.
  • the structural coloring body 23 may be either an inorganic substance or an organic substance, but is preferably an organic substance because it can be easily bonded to both the support substrate 21 and the nucleic acid ligand 13.
  • a helical organic substance is suitably used.
  • the helical organic substance include proteins or polypeptides having a helical structure, polysaccharides, and DNA.
  • the proteins include ⁇ -keratin, myosin, evidamine
  • the polypeptide a polypeptide having an ⁇ -helical structure is suitably used.
  • Amylose that forms a helical structure is suitably used as the polysaccharide.
  • proteins or polypeptides are preferably used because it is easy to give selectivity to the binding to the support substrate 21 and the nucleic acid ligand 13 depending on the type of constituent amino acids (side chains). Furthermore, as these helical organic substances, artificially prepared polypeptides are most preferably used because the binding selectivity to the support substrate 21 or the nucleic acid ligand 13 can be easily optimized.
  • the length of the structural color body 23 in the longitudinal direction is preferably 3 to 1500 nm because an ⁇ -helical structure is easily formed while considering the limit of polymerization while maintaining the secondary structure. More preferably, it is 300-810 nm. When detecting with visible light, it is preferable that the wavelength is within the range of the wavelength of visible light (for example, 400 to 800 nm).
  • the lateral length of the structural chromophore 23 is preferably 0.8-2. Onm, which is mainly determined by the type of constituent amino acids.
  • the test chip 11 shown in FIGS. 1 and 3 has a support substrate 21 whose surface or the whole is made of an inorganic material, and a bonding group 22 for covalent bonding to the support substrate 21 (an alkyl group and a silane group in FIG. 3).
  • a structural chromophore 23 comprising a polypeptide having As the inorganic material, glass or quartz capable of holding a hydroxyl group (—OH) is preferably used, and silicon or silica is particularly preferably used.
  • the structural coloring body 23 is formed by removing the protecting group 24 from the precursor 23a having the bonding group 22 and the protecting group 24 (in FIG. 3, ⁇ -benzyloxycarbol group). You.
  • the bonding group 22 can be covalently bonded to an inorganic material constituting the surface of the support substrate 21.
  • the bonding group 22 include a functional group that functions as a coupling agent such as a silane coupling agent.
  • the coupling agent has an organic functional group and a hydrolyzable group in one molecule.
  • silane-terminated polyallylamines as shown in FIG. 3 are used. That is, examples of the organic functional group include an alkyl group and the hydrolyzable group. And a silane group.
  • the silane group is hydrolyzed by water to form a silanol, and then the silanols partially condense into an oligomer-like state, and form a hydrogen bond with the hydroxyl group (1-OH) of the inorganic material. Is adsorbed. Subsequently, the silanol adsorbed on the inorganic material is dehydrated and condensed by being heated to a predetermined temperature, and forms a strong covalent bond as shown in FIG. Further, the silanols which have become the oligomer-like state due to the heating also undergo dehydration condensation, and are covalently bonded so that the spacing between adjacent silanols, that is, the spacing between adjacent precursors 23a is constant below a specific light wavelength.
  • This binding group 22 is located at the end of the structural chromophore 23.
  • the structural color body 23 for example, as shown in FIG. 3, a structure is formed in which an alkyl group connects between the terminal of the polypeptide and the silane group.
  • the structural chromophore 23 (polypeptide having the bonding group 22) contains at least basic amino acids as constituent amino acids, and it is preferable that all of the constituent amino acids are basic amino acids.
  • Basic amino acids include lysine, arginine or histidine. Each of these basic amino acids has a positive charge under predetermined conditions and participates in electrostatic binding to the above-described nucleic acid ligand 13 having a negative charge. That is, the polypeptide constituting the structural chromophore 23 must contain an amino acid other than the basic amino acid, and even if it has a positive charge as a whole. For this reason, the number of basic amino acids must exceed the number of acidic amino acids.
  • basic amino acid lysine or arginine which surely has a positive charge in the vicinity of PH6-8 where the nucleic acid ligand 13 easily forms a higher-order structure is preferably used.
  • these basic amino acids can be used in both L-form and D-form.
  • the protective group 24 is not present in the structural color former 23 but is present only in the precursor 23a.
  • This protecting group 24 is dissolved in the reaction solution as a monomer when preparing the precursor 23a, and does not take part in the side chain (positive charge) of the basic amino acid in the polymerization reaction. And play a protective role.
  • the precursor 23a synthesized by the chemical polymerization reaction is linearly polymerized only by the peptide bond, so that an appropriate ⁇ -helix structure is easily formed.
  • Examples of the protecting group 24 include an ⁇ -amino group on the side chain of lysine, arginine or histidine, Those which react selectively with an azyl group or an imidazole group and which do not react with an amino group directly bonded to the carbon atom at the a position of the same amino acid are used.
  • a solvent having low reactivity with an amino acid such as dimethylformamide (DMF) is preferably used.
  • the protecting group 24 is removed from the precursor 23a by a hydrolysis treatment using a hydrolytic reagent such as hydrogen bromide (HBr) Z acetic acid (AcOH). That is, the protective group 24 is removed from the precursor 23a by performing the hydrolysis treatment, and the precursor 23a becomes the structural color body 23 (in this case, the structural color body 23 does not include the bonding group 22).
  • a hydrolytic reagent such as hydrogen bromide (HBr) Z acetic acid (AcOH).
  • the protective group 24 is removed from the precursor 23a by performing the hydrolysis treatment, and the precursor 23a becomes the structural color body 23 (in this case, the structural color body 23 does not include the bonding group 22).
  • the precursor ratio of the precursor 23a may be 1 or less
  • the aspect ratio of the structural color body 23 needs to exceed 1 as described above. That is, the structural color body 23 may have an aspect ratio exceeding 1 only by removing the protecting group 24 from the precursor 23a.
  • the method for manufacturing the inspection chip 11 includes a film forming step of forming a monomolecular film 25 by bonding the precursor 23a to the surface of the support substrate 21 with a bonding group 22, and forming the film in the film forming step.
  • the monomolecular film 25 formed in the film forming step refers to a thin film of the precursor 23a bonded to the surface of the support substrate 21 via the bonding group 22.
  • the precursor 23a is added to water or an aqueous solution such as a buffer solution using water as a solvent, and the precursor 23a is floated so as to cover the entire water surface. After transferring 23a almost as it is onto the surface of the support substrate 21 and transferring it, the precursors 23a are fixed to the surface of the support substrate 21. As a result, a protective group-bonded substrate 12a as shown in FIG. 1 (a) is produced.
  • the method of transferring the precursor 23a floating on the water surface onto the surface of the supporting substrate 21 is performed, for example, according to the Langmuir-Blodgett method (LB method) described in Patent Documents 1 and 2 described above. Is
  • the silane group located at the end of the bonding group 22 bonded to each precursor 23a is hydrolyzed to silanol, and then the silanol group adjacent to the water surface is decomposed.
  • the polymer is partially condensed by hydrogen bonding to form an oligomer-like state and float.
  • the silanol located at the end of the binding group 22 bound to each precursor 23a is converted to the support substrate 21.
  • a thin film-like monomolecular film 25 in which a number of precursors 23a are arranged vertically and horizontally adjacently at a density equal to or less than the wavelength of a specific light is formed on the surface of the supporting substrate 21, a thin film-like monomolecular film 25 in which a number of precursors 23a are arranged vertically and horizontally adjacently at a density equal to or less than the wavelength of a specific light is formed.
  • the supporting substrate 21 to which the monomolecular film 25 has been adsorbed is heated to a predetermined temperature while drying, so that the monomolecular film 25 is fixed on the supporting substrate 21 by a covalent bond to form a protective group bonding type.
  • a substrate 12a is obtained.
  • each precursor on the protective group-bonded substrate 12a is hydrolyzed by allowing a hydrolysis reagent such as HBrZAcOH to act on the surface of the protective group-bonded substrate 12a.
  • the protecting group 24 is separated from 23a.
  • the monomolecular film 25 on the surface of the protective group-bonded substrate 12a has a positive charge, and the structural coloring substrate 12 is obtained.
  • the binding step the nucleic acid ligand 13 having a negative charge is electrostatically bound to the surface of the monomolecular film 25 having a positive charge.
  • This binding step is most preferably performed under binding conditions such that the higher-order structure of the nucleic acid ligand 13 is maintained as it is.
  • the binding condition is preferably a condition under which the target 14 specifically binds to the nucleic acid ligand 13, more preferably an environmental condition in which the test mixture was placed, or a physiological condition as described above.
  • the binding condition may be a condition in which at least one selected from the group consisting of PH , salt concentration, and temperature is milder than the environmental condition or physiological condition.
  • the above-mentioned mild conditions refer to conditions that hardly affect the intramolecular hybridization of the nucleic acid ligand 13.
  • Such binding conditions include, for example, setting the pH of the solution within the range of 6-8, and setting the salt concentration to be equal to or lower than physiological saline.
  • the temperature is more preferably 0 ° C. and 50 ° C. or less, more preferably 10-40 ° C., and even more preferably 20-37 ° C.
  • this test chip 11 When using this test chip 11 to test the target 14 contained in the test mixture, first, a sample solution containing the test mixture is added to the surface of the test chip 11 and reacted for a predetermined time. At this time, if the target 14 is contained in the sample solution Then, the target 14 specifically binds to the nucleic acid ligand 13 on the surface of the test chip 11. Next, the surface other than the target 14 is removed from the surface of the inspection chip 11 by washing the surface of the inspection chip 11 after the reaction with the sample liquid. At this time, only the target 14 is present on the surface of the test chip 11 while maintaining specific binding.
  • the presence or absence of the target 14 contained in the sample liquid is detected by visually or by an inspection device detecting a change in the structural coloring on the surface of the inspection chip 11 before and after the reaction.
  • a spectrophotometer is suitably used as the inspection device. This spectrophotometer measures the reflectance of light on the surface of the inspection chip 11 for a continuous, predetermined range of wavelengths (for example, 300 to 700 nm as shown in FIG. 4), thereby obtaining visible light, infrared light, or ultraviolet light. The reflection spectrum of is analyzed.
  • an inspection device other than the spectrophotometer for example, an atomic force microscope (AFM topography image) is used.
  • AFM topography image As shown in Fig. 2 (d), the part where the target 14 specifically bound was whiter than the surrounding color, and the height difference of the surface changed dramatically. You can see that there is. Therefore, in the inspection method using the AFM topography image, it is possible to detect whether or not the target 14 exists in the sample liquid. Further, as shown in FIG. 2 (d), it is possible to accurately count the number of targets 14, so that the inspection can be performed in more detail and the quantification is excellent. However, when performing a simple inspection in a short time, it is better to measure with a spectrophotometer.
  • the light reflectance is measured by the spectrophotometer, at least a shift (change) in the reflectance is clearly confirmed by the coupling between the inspection chip 11 and the target 14. It is sufficient to measure the reflectance at one specific wavelength (eg, 353 nm reflectance) or the reflectance spectrum over a continuous narrow range of wavelengths (eg, 350-400 nm). In this case, it is not necessary to check the wavelengths in a continuous and wide range, and the operation is easily and quickly performed. Further, at this time, since a change in reflectance specific to the binding of the target 14 is detected, the possibility of detecting non-specific binding due to factors other than the target 14, that is, noise, is easily reduced. .
  • the present embodiment has the following advantages.
  • the test chip 11 includes a structural coloring substrate 12 and a nucleic acid ligand 13 bound to the structural coloring substrate 12. Further, the nucleic acid ligand 13 is composed of a nucleic acid having a higher-order structure. That is, the test chip 11 tests the presence or absence of the target 14 based on the specific binding between the nucleic acid ligand 13 having a higher-order structure and the target 14 having a three-dimensional structure. Inspection accuracy and reliability are extremely high. Further, in the inspection chip 11, since the structural coloring substrate 12 itself exhibits structural coloring, the presence of the target 14 is detected by the target 14 partially blocking the structural coloring. Therefore, the labor and time required for the inspection can be reduced, and extremely simple and quick inspection can be performed.
  • the nucleic acid ligand 13 obtained by the SELEX method requires more time and effort than the production of antibodies used in immunological techniques such as the ELISA method, in particular, monoclonal antibodies that can increase the test accuracy. It is easy to make the time extremely short.
  • the ELISA test requires many steps and time, such as binding a secondary antibody bound to an enzyme involved in color development to an antigen, and further performing a reaction for color development and its detection.
  • the test method of the present embodiment does not require a secondary antibody, so that the test can be performed extremely simply and quickly.
  • the operation of the nucleic acid ligand 13 is simple and inexpensive in comparison with an antibody because an animal does not need to be used for its production.
  • it can be used for antibodies that are difficult to produce, such as those that are toxic to animals and those that are easily degraded in the bloodstream.
  • the test method of the present embodiment does not require the above-described DNA extraction and expensive equipment.
  • the inspection method using the inspection chip 11 it is possible to shorten the inspection period until the product is shipped, and it is possible to carry out the simple siding by the kit siding. Yes The absence can be visually confirmed.
  • this test method is superior to the ELISA method and the PCR method in that the test can be performed using visible light at the place where the sample solution is collected and prepared.
  • the method for manufacturing the inspection chip 11 includes a film forming step of forming a monomolecular film 25 of the precursor 23 a (the structural color former 23 having the protective group 24) on the surface of the support substrate 21. And a binding step of binding the nucleic acid ligand 13 to the positively charged monomolecular film 25 by removing the protecting group 24 from the monomolecular film 25 thus obtained. Therefore, it is possible to extremely easily manufacture the inspection chip 11 that can easily, quickly, and accurately inspect the target 14 contained in the test mixture. In particular, in this production method, since the monomolecular film 25 is positively charged and binds to the negative charge of the nucleic acid ligand 13, the three-dimensional structure of the nucleic acid ligand 13 is hardly affected in the binding step. Therefore, the inspection accuracy can be easily increased.
  • the thickness of the SiO layer can be controlled by the treatment conditions.
  • the structural coloring substrate 12 of a desired color can be simply produced. At this time, it is very convenient because it can be adjusted to a structural coloration convenient for detection.
  • the structural coloring substrate 12 produced during the production of the test chip 11 can bind not only the nucleic acid ligand 13 but also a protein or polysaccharide having a negative charge.
  • a coupling agent such as a titanium coupling agent may be used.
  • the target molecule may be a nucleic acid such as DNA or RNA.
  • the nucleic acid binds to the nucleic acid ligand 13 in a higher-order structure-dependent manner without binding to the nucleic acid ligand 13 through hybridization depending on the primary structure.
  • the structural color-developing substrate 12 that exhibits structural coloration by fixing the structural color-developing body 23 to the surface of the support substrate 21 was manufactured. Meanwhile, the surface of the support substrate 21 made of silicon may be baked in the presence of oxygen to form an oxide layer, thereby producing the structural coloring substrate 12 exhibiting structural coloring. At this time, the inspection chip 11 Nucleic acid ligands 13 that are not interposed via structural color formers 23 are bound to the surface of a positively charged support substrate 21 having an oxide layer.
  • ⁇ -benzyloxycarbonyl-L-lysine (BCL) d-aminopropyltnmetnoxysilane The polymerization reaction was performed in a (DMF) solvent for 24 hours at 25 ° C.
  • the degree of polymerization was measured by 1 H-NMR.
  • the confirmation of the formation of the ⁇ -helix constituting the secondary structure and the measurement of the content were performed using a Circular Dichorism spectroscope.
  • the absorption peaks of amide I and amide II were 1652 cm- 1 and 1543 cm- 1 , respectively, confirming that the silane-terminated PBCL formed an ⁇ -helical structure.
  • the ⁇ -helix content of the silane-terminated PBCL dissolved in trifluoroethanol was 60%.
  • the support substrate 21 of silica (SiO 2) is formed.
  • silane-terminated PBCL solution was floated on a surface of pure water at 25 ° C. so as to be spread in a single layer.
  • Such a silane-terminated PBCL solution was transferred onto the support substrate 21 using the LB method so that the surface pressure became a constant pressure of approximately 5 mNZm.
  • a silane-terminated PBCL monomolecular film 25 was deposited on the surface of the support substrate 21 in a thin film form.
  • the silane-terminated PBCL is covalently bonded to the surface of the support substrate 21 to form a protective group.
  • a mold substrate 12a was produced.
  • the silane-terminated PBCL The self-assembly of the ⁇ -helix in a single layer can easily be estimated from the AFM topography image in Fig. 2 (a).
  • the silane-terminated PBCL force is also reduced to the ⁇ -benzyloxycarbonyl group ( ⁇ -benzyloxycarbonyl) as the protective group 24. group) has been removed.
  • the monomolecular film 25 of poly (L-lysine) (PLL) having a positive charge is fixed on the surface of the support substrate 21 in a thin film shape.
  • PLL poly (L-lysine)
  • the change in the thickness and the degree of wetting of the monomolecular film 25 of the structural color body 23 can be seen from the same image.
  • the surface of the structural coloring substrate 12 on which the monomolecular film 25 is fixed in a thin film form has a force that does not show any difference in color development as compared with that before the monomolecular film 25 is fixed (visually). Confirmation).
  • RNA aptamer consisting of the base sequence represented by SEQ ID NO: 1 (nucleic acid ligand 13 also shown in the lower right part of FIG. 3) is electrostatically charged with respect to the positive charge of the PLL.
  • an aptamer chip as the inspection chip 11 was produced.
  • the AFM topography image of this aptamer chip is shown in Fig. 2 (c).
  • the RNA aptamer was selected by the SELEX method as specifically binding to the cell membrane surface of Sphingobium yanoikuyae. This RNA abtamer is fixed to the structural coloring substrate 12 in a state where a stem-loop structure as a higher-order structure is formed.
  • the maximum value of the reflectance decreased from about 83% (curve b in FIG. 4: 353 nm) to about 68% (curve a in FIG. 4: 388 nm). That is, in this aptamer chip, the structural coloring was partially blocked by contact with the Sphingobiumyanoikuyae fungus. Further, in the AFM topography image of FIG. 2 (d), the site where the Sphingobiumyanoikuyae was bound appeared white, and contact with the Sphingobium yanoikuyae was confirmed. From the above, the presence of the target 14 in the sample could be easily and quickly inspected by using the inspection chip 11 which also has a thread and alignment force between the RNA abtamer and the structural coloring substrate 12.
  • the reflection spectrum at a wavelength of 300 to 700 nm was examined. For example, by examining only the shift amount of the reflectance at a specific wavelength such as 353 nm or 388 nm, the aptamer spectrum was determined. Binding of target 14 to the chip is detectable. Similarly, the binding of the target 14 to the aptamer chip can be detected by examining only the shift amount of the reflection spectrum in a specific continuous range of, for example, 350 to 400 nm.

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Abstract

L'invention concerne une puce d'inspection (11) qui comprend un substrat chromogène structurel (12), et des ligands d'acides nucléiques (13) liés audit substrat. Les ligands d'acides nucléiques (13) sont constitués respectivement d'un acide nucléique de structure de poids fort, tel qu'un aptamère d'ARN. Ledit substrat chromogène structurel (12) comporte une plaque de support (21) et, fixé sur une surface associée, un film monomoléculaire (25) du chromogène structurel (23). Ledit chromogène structurel (23) est, de préférence, constitué d'un polypeptide contenant des acides aminés basiques. Dans cet état, le film monomoléculaire (25) du substrat chromogène structurel (12) présente des charges positives de manière à pouvoir engendrer une liaison électrostatique avec les ligands d'acides nucléiques (13).
PCT/JP2004/014408 2003-10-01 2004-09-30 Puce d'inspection, procede de production associe et procede d'inspection au moyen de ladite puce WO2005033702A1 (fr)

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JP2003-343732 2003-10-01
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JP2004153942 2004-05-24

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2002345494A (ja) * 2001-03-23 2002-12-03 Fuji Photo Film Co Ltd ハイブリダイゼーションプローブ及びそれを用いた標的核酸検査キット、標的核酸検査装置、標的核酸検査方法
JP2003043037A (ja) * 2001-07-27 2003-02-13 Inst Of Physical & Chemical Res ハイブリダイゼーション用基板、この基板の製造方法及び使用方法
JP2003222629A (ja) * 2002-01-31 2003-08-08 Fuji Photo Film Co Ltd リセプター・リガンド会合反応方法およびそれに用いるリアクタ

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2002345494A (ja) * 2001-03-23 2002-12-03 Fuji Photo Film Co Ltd ハイブリダイゼーションプローブ及びそれを用いた標的核酸検査キット、標的核酸検査装置、標的核酸検査方法
JP2003043037A (ja) * 2001-07-27 2003-02-13 Inst Of Physical & Chemical Res ハイブリダイゼーション用基板、この基板の製造方法及び使用方法
JP2003222629A (ja) * 2002-01-31 2003-08-08 Fuji Photo Film Co Ltd リセプター・リガンド会合反応方法およびそれに用いるリアクタ

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