WO2015170711A1 - プラズモニックチップおよびそれを用いるがん疾病の蛍光画像ならびにラマン分光による診断方法 - Google Patents
プラズモニックチップおよびそれを用いるがん疾病の蛍光画像ならびにラマン分光による診断方法 Download PDFInfo
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- G01N21/64—Fluorescence; Phosphorescence
- G01N21/645—Specially adapted constructive features of fluorimeters
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- G01N21/6458—Fluorescence microscopy
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- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
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- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/645—Specially adapted constructive features of fluorimeters
- G01N21/648—Specially adapted constructive features of fluorimeters using evanescent coupling or surface plasmon coupling for the excitation of fluorescence
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- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
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- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
- G01N33/57473—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving carcinoembryonic antigen, i.e. CEA
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- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
- G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
Definitions
- the present invention enhances autofluorescence and tumor markers by localized surface plasmon resonance by capturing a fluorescence observation substrate (plasmonic chip) suitable for fluorescence imaging diagnosis with a fluorescence microscope and a body fluid, particularly a cancer-related substance in blood.
- the present invention relates to a method for diagnosing cancer diseases by using fluorescence images and by Raman spectroscopy.
- methylated DNA as a cancer-related substance and histone-binding proteins (nucleosomes and chromatin) bound to them are attracting attention, and they are captured and crystallized or aggregated. It is important for simple diagnosis and early detection of cancer presence to analyze the methylation state of DNA and the chemical modification state of histone code by irradiating the crystal with laser.
- DNA is composed of unique sequences of four bases, adenine (A), guanine (G), thymine (T) and cytosine (C). These bases are A and T and G and C pairs on the two strands forming a DNA double helix. These pairs of chains store information for making specific molecules grouped into regions called genes. Within each cell there is a process that controls which genes are turned on, ie, expressed, to define the cell's unique function. One of these control mechanisms is provided by adding a methyl group to cytosine (C). C labeled with a methyl group can be expressed as mC.
- A adenine
- G guanine
- T thymine
- C cytosine
- DNA methylation is said to play an important role in determining whether some genes are expressed, and by turning off genes that are not needed, DNA methylation It is an indispensable control mechanism for function. Or abnormal DNA methylation is said to be one of the mechanisms underlying the changes observed with aging and the development of many cancers.
- Cancer has traditionally been linked to genetic changes caused by chromosomal mutations in DNA. Mutations, whether inherited or acquired, can result in loss of expression of genes that are critical to maintaining a healthy state. It is now supported that a relatively large number of cancers are often caused by inappropriate DNA methylation that is close to DNA mutations. In many cases, DNA hypermethylation turns off critical genes, such as tumor suppressor genes or DNA repair genes, allowing cancer to develop and progress. This non-mutation process for controlling gene expression is called epigenetic.
- DNA methylation is a chemical modification of DNA in which a methyl group (m) is added to a specific cytosine (C) of DNA, which is performed by an enzyme called methyltransferase.
- This non-mutation (epigenetic) process (mC) was said to be a critical factor in the regulation of gene expression (Non-patent Document 1).
- mC non-mutation (epigenetic) process
- SPR surface plasmon resonance method
- Patent Document 2 Patent Document 2
- ELISA enzyme immunosorbent assay
- Patent Document 3 a method of using a plasmonic chip by forming a thin film of silver and zinc oxide on a substrate having a periodic structure with a pitch of 350 nm as a plasmonic chip that realizes high-level detection, sensitivity, and rapidity of a fluorescent label marker.
- Patent Document 3 metal nanoparticles dispersed in an organic solvent, the organic solvent is volatilized, and the metal nanoparticles are self-organized in a two-dimensional direction to form a localized plasmon fluorescence enhancing sheet composed of silver nanoparticles with a uniform grain system.
- the present invention provides a plasmonic chip that can be easily mass-produced, and realizes adsorption of an adsorbing substance such as a cancer-related substance or an antibody or adsorption of a fluorescently labeled marker to the plasmonic chip.
- a first object is to provide a plasmonic chip capable of enhancing fluorescence emission by several to several tens of times by enhancement and diagnosing a fluorescence image, and a method for diagnosing cancer diseases using the same.
- a silver complex quantum crystal or a silver peroxide mesocrystal substrate formed by subjecting the silver complex quantum crystal to an alkali treatment is irradiated with light, including UV and infrared light.
- silver peroxide mesocrystals can utilize the physical phenomenon of localized surface plasmon resonance (having a localized surface plasmon resonance effect), as well as secondly when forming a silver complex quantum crystal
- it has a chemical property capable of making a chip that adsorbs an antibody, a fluorescently labeled marker, or a fluorescently labeled marker that binds to a ligand.
- silver peroxide mesocrystals have a chemical property of aggregating cancer-related substances including nucleosomes containing DNA in serum or chromatin, particularly histones wound with DNA. Therefore, according to the first aspect of the present invention, by using such a plasmonic chip, nucleosome or chromatin is selectively captured as a tumor marker or a cancer-related substance, and fluorescence imaging is performed with a normal fluorescence microscope to determine whether or not cancer exists.
- the present invention has been made by finding that it is possible to determine the fluorescence image of the present invention, which has a silver complex quantum crystal region, adsorbs a tumor marker together with a cancer-related substance, and is enhanced by a localized surface plasmon resonance function
- a localized surface plasmon resonance function For observing autofluorescence images of plasmonic chips and silver complex nanocrystals containing silver peroxide, which are obtained by alkali treatment of silver complex quantum crystals, and enhanced cancer-related substances with localized surface plasmon resonance function Fluorescence image observation that has an enhanced mesocrystal region, captures adsorbents such as cancer-related substances, and is enhanced by the localized surface plasmon resonance function There are plasmonic chips for.
- a second aspect of the present invention is a method for diagnosing cancer diseases using the plasmonic chip, one of which is a silver oxide containing silver peroxide obtained by subjecting a silver complex quantum crystal to an alkali treatment.
- Fluorescence image diagnosis method for cancer disease characterized by irradiating light to crystal region adsorbed and aggregating on plasmonic chip, and diagnosing presence or absence of cancer by performing fluorescence image diagnosis by autofluorescence of cancer-related substance It is.
- a tumor marker having a fluorescent function is combined with the ligand of the metal complex quantum crystal or of the ligand.
- the position of cancer-related substances aggregated on the substrate cannot be determined visually. Therefore, a method for diagnosing cancer characterized in that the position of the crystal region is determined by microscopic image diagnosis of a microscope, and the laser is irradiated to the determined crystal to analyze histone tail chemical modification and remodeling factors.
- the third aspect of the present invention is that Raman spectroscopy is performed by irradiating various laser beams of 514, 532, 633, and 785 nm with an image including a fluorescence image of a cancer-related substance captured on a plasmo chip as a mark. The purpose is to make a precise analysis.
- the present invention selectively captures cancer-related substances in body fluids, particularly blood, analyzes the structure of histones or chromatin, and analyzes the factors that chemically modify histone tails, thereby determining the presence or absence of cancer. It begins and enables early diagnosis of cancer.
- a cancer-related substance or a tumor marker can be selectively adsorbed, so that the autofluorescence of the cancer-related substance or the fluorescence intensity of the tumor marker is localized on the surface. Fluorescence image determination can be facilitated by enhancement by plasmon resonance.
- the present inventors agglomerate quantum crystals (nano-sized metal complex crystals) by chemically reducing an aqueous metal complex solution on a metal substrate that has a lower base potential than the metal forming the complex (which tends to be ionized) due to the electrode potential difference. I am letting.
- a silver thiosulfate aqueous solution is agglomerated on copper or a copper alloy having a lower electrode potential (higher ionization tendency) than silver, thereby forming a quantum complex of the silver complex using a chemical reduction method.
- the concentration of the metal complex in the aqueous solution should be determined mainly in consideration of the size of the quantum crystal to be formed, and when using a dispersant, the concentration should also be taken into account, and is usually from 100 ppm to 5000 ppm. However, depending on the function of the ligand, a concentration of 500 to 2000 ppm is preferable for preparing a nanosize that should be called a nanocluster.
- the metal complex forming the quantum crystal is selected so as to have a complex stability constant (log ⁇ ) or more represented by the formula (I) correlated with the electrode potential E of the supported metal.
- E ° (RT /
- E ° is a standard electrode potential
- R is a gas constant
- T is an absolute temperature
- Z is an ion valence
- F is a Faraday constant.
- the metal complex is a plasmon metal complex selected from Au, Ag, Pt or Pd, it has a localized surface plasmon resonance enhancing effect on Raman light.
- the metal complex when it is a silver complex, it is preferably formed by a reaction between a silver complexing agent having a stability constant (generation constant) (log ⁇ i) of 8 or more and silver halide.
- a silver complexing agent having a stability constant (generation constant) (log ⁇ i) of 8 or more
- silver chloride is preferred, and the complexing agent is preferably one selected from thiosulfate, thiocyanate, sulfite, thiourea, potassium iodide, thiosalicylate, and thiocyanurate.
- the silver complex has quantum dots composed of nanoclusters having an average diameter of 5 to 20 nm, and the size of the quantum crystal is 100 to 200 nm.
- the silver oxide composite needle-like nanocrystal group of the present invention is such that a silver oxide containing silver peroxide is self-assembled to form a neuron-like three-dimensional superstructure (mesocrystal) (FIGS. 8 and 9).
- Silver ion aqueous solution can be formed by performing constant potential electrodeposition using an Ag / AgCl electrode, or by oxidizing silver quantum crystals by alkali treatment, but silver complex quantum crystals such as silver thiosulfate It can be easily formed by subjecting the quantum crystal to an alkali treatment (treatment with a sodium hypochlorite aqueous solution).
- a plasmonic chip of the present invention it is possible to determine the presence or absence of a cancer-related substance and a chemical modification factor that controls chromatin remodeling in a biological sample contained in blood by fluorescent image diagnosis.
- a plasmonic chip having a silver halide or silver oxide-containing composite needle-shaped nanocrystal group, that is, a mesocrystalline region of silver oxide containing silver peroxide (FIGS. 8 and 9) is prepared. Serum or a biological sample solution is dropped on the needle-shaped nanocrystal group region of the monic chip to selectively capture a cancer-related substance having a positive charge in the sample.
- Serum cancer-related substances are thought to include histones (nucleosomes) in which DNA is wound once, and chromatin (fibers) in a structure in which they are gathered into a string. Globulin is positively charged, but its increase is up to 2 times less than other cancer-related substances, so the substance detected by the present invention increases more than 100 times as cancer progresses. It shows that an increase (cancer-related substance) other than is detected. Further, DNA from normal cells, DNA acetylated and dissociated from histones, and albumin occupy about 60% of serum, but are negatively charged and are not adsorbed in the present invention. Therefore, it is convenient for fluorescence imaging diagnosis of cancer-related substances.
- FIG. 15 shows 100-fold, 3000-fold, and 3D images of 10-fold and 100-fold dilutions of benign disease, gastric cancer, and colon cancer.
- Fig. 16 shows KatoIII (Gastrointestinal Ring Cell Carcinoma) cultured and observed by diluting double-stranded DNA, RNA and protenes extracted from the cancer cells.
- (A, b, c) use a laser microscope (D, e, f) is the DNA extracted from it, (g, h, i) is the RNA, and (j, k, l) is the surface structure of proten 100x, 3000x (image and undulation) showing 3D image, (m, n, o) is 100x, 1000x showing the surface structure when cancer cells are physically crushed (image and undulation) Undulation) shows a 3D image.
- D, e, f is the DNA extracted from it
- (g, h, i) is the RNA
- (j, k, l) is the surface structure of proten 100x, 3000x (image and undulation) showing 3D image
- (m, n, o) is 100x, 1000x showing the surface structure when cancer cells are physically crushed (image and undulation) Undulation) shows a 3D image.
- FIG. 17 shows chemically cultured cancer cells (a), DNA (b) extracted therefrom, RNA (c), protene (e), physically crushed cancer cells (e), stomach cancer
- Figure 6 shows Raman scattering spectra of sera from patients (f), sera from colon cancer patients (g), and sera from patients with benign disease (h) on a mesocrystal proteonic chip.
- KatoIII stomach ring cell carcinoma
- MKN-45 poorly differentiated gastric cancer
- CW-2 Japanese common colorectal cancer cell line
- PK45-P human pancreatic cancer cell line
- NHDF-Neo skin fibroblasts
- the acicular nanocrystals (silver oxide mesocrystals containing silver peroxide) of the present invention are likely to be negatively charged in an aqueous solution because the silver oxide containing silver peroxide is in contact with the sample (target molecule). It seems to form a charge transfer complex. Further, since silver oxide is reduced by receiving light energy and deposits metallic silver, it has an enhancing effect due to localized surface plasmon resonance possessed by regularly arranged metal nanoparticles.
- the needle-shaped nanocrystal (mesocrystal) of the present invention is a non-metal, but has both metal properties and ionization properties, so that it becomes a plasmonic chip suitable for surface enhanced Raman scattering (SERS) measurement, and as a cancer diagnostic chip. Convenient.
- Quantum crystals formed on metal substrates or metal particles are likely to have positive polarity in aqueous solution as metal complex crystals, and in order to adsorb and fix proteins in biological samples, they are alkaline in the presence of halogen ions. It is preferable to adjust the polarity by dropping a sodium hypochlorite aqueous solution having a pH of 11 or more. Quantum crystals are not only recrystallized and become negative in aqueous solution, but silver oxide composite needle-shaped nanocrystals form peroxides. Can be promoted.
- crystallization of the cancer related substance of gastric cancer adsorbed on the plasmonic chip is shown.
- a fluorescence micrograph of a red laser of a cancer-related substance crystal of gastric cancer adsorbed on a plasmonic chip is shown.
- crystallization of the cancer related substance of the stomach cancer adsorbed on the plasmonic chip is shown.
- a fluorescence micrograph of a cancer-related substance crystal of colorectal cancer adsorbed on a plasmonic chip is shown.
- Figure below The XPS measurement result of the alkali-treated recrystallized substrate is shown.
- the XPS measurement result after etching the surface of a recrystallized substrate is shown.
- a photomicrograph of a cancer-related substance crystal adsorbed on a plasmonic chip is shown. (A) shows benign disease, (b) shows stomach cancer, and (c) shows colon cancer.
- a RGB micrograph is shown in a laser micrograph of a serum-adjusted sample obtained from a benign disease patient.
- the RGB micrograph is shown in the laser micrograph which made the sample which adjusted the serum obtained from the colon cancer patient.
- the Raman spectrum which shows the state of the chemical modification of the histone tail which is a gastric cancer related substance is shown.
- samples were diluted 10-fold and 100-fold benign diseases (a, b, c; d, e, f), gastric cancer (g, h, i) ; j, k, l), 100 ⁇ , 3000 ⁇ , 3D image of (m, n, o; p, q, r) of colon cancer (Colon cancer).
- a 10-fold diluted benign disease (s, t), gastric cancer (u, v), colon cancer (w, x) (w, x) can be obtained using a 3D laser scanning microscope.
- each corresponding RGB color histogram of the resulting image (3000 times) is shown.
- (A, b, c) is KatoIII (stomach ring cell carcinoma) using a laser microscope
- (d, e, f) is DNA extracted therefrom
- (g, h, i) is RNA
- ( j, k, l) shows the surface structure of proten, 100x, 3000x (image and undulation)
- 3D image shows the surface when cancer cells are physically crushed 100x, 1000x (image and undulation) showing the structure
- 3D image is shown.
- Chemically cultured cancer cells (a), extracted DNA (b), RNA (c), protene (e), physically crushed cancer cells (e), from stomach cancer patients 2 shows the Raman scattering spectrum of the serum (f) in the 1200-1600 nm region on the mesocrystal proteonic chip of serum (g) from a colon cancer patient and serum (h) from a patient with benign disease.
- KatoIII stomach ring cell carcinoma
- MKN-45 low-differentiated gastric cancer
- CW-2 Japanese common colorectal cancer cell line
- PK45-P human pancreas
- NHDF-Neo indicates (skin fibroblast).
- a histone-related antibody diagram for identifying histone tail chemical modifications is shown (www.genetex.com). It is a fluorescence image which shows the benign disease observed on the plasmonic chip of this invention, and the autofluorescence state of cancer.
- Example 1 A 1000 ppm aqueous solution of silver thiosulfate was prepared, one drop was dropped on a phosphor bronze plate, allowed to stand for about 3 minutes, and the solution was blown away. As a result, an SEM image produced quantum crystals.
- the figure is a photograph showing various SEM images of the nanoparticle aggregate (quantum crystal) produced in Example 1, which is a thin hexagonal columnar crystal of about 100 nm, and has irregularities on the order of several nm. No facets specific to metal nanocrystals could be confirmed.
- FIG. 6 is a photograph showing the relationship between the standing time after dropping on the phosphor bronze hill and the quantum crystal shape.
- FIG. 4 is a graph showing the results of EDS spectrum (elemental analysis) of the quantum crystal.
- the crystals formed on the phosphor bronze plate detected elements derived from silver and complex ligands, but prepared a 1000 ppm aqueous solution of silver thiosulfate on the copper plate, dropped one drop and left it for about 3 minutes. When it was blown away, only silver was detected.
- the equilibrium potential of the silver thiosulfate complex is 0.33 and the copper Since it is equivalent to the electrode potential (0.34), only silver (0.80) is deposited on the copper substrate, and in the case of phosphor bronze, the electrode potential is slightly base, 0.22, so that the silver complex It is probable that the crystals were precipitated. Therefore, in order to prepare a quantum crystal, 1) the complex aqueous solution is a dilute region of 500 to 2000 ppm, and 2) the electrode potential of the supported metal is slightly lower than the equilibrium potential of the metal complex aqueous solution. ) It seems to be important that the metal complex is aggregated by the electrode potential difference. The same was true when a 1000 ppm thiourea silver complex aqueous solution was used.
- the peak near 529 eV is an O peak derived from silver peroxide (AgO)
- the peak near 530 eV is silver oxide (Ag 2 O). Origin O Recognized as a peak.
- the O peak derived from silver peroxide (AgO) in the peak near 529 eV is larger than the O peak derived from silver oxide (Ag2O) in the peak near 530 eV. It can be said that silver peroxide is formed in the vicinity of the substrate. This is presumed to be due to the catalytic action during mesocrystal formation and the electrode potential of the substrate.
- the EDS measurement was performed using the above-mentioned recrystallized substrate model: JEOL Ltd./JSM-7001F (field emission analysis scanning electron microscope). Further, even when the silver thiosulfate quantum crystals were treated with a hypochlorous acid aqueous solution, a 0.01 N sodium hydroxide aqueous solution, a 0.01 N hydrochloric acid aqueous solution, and a 0.1 molar sodium carbonate aqueous solution, similar results were not obtained. Therefore, it seems that the formation of the needle-like crystals is caused by the above reaction in the presence of silver ions and thiosulfate ions. Silver oxide is negatively charged in an aqueous solution and is reduced by light to precipitate metallic silver. Since silver peroxide has such a tendency, it is considered that a positively charged cancer-related substance is adsorbed, and a surface plasmon enhancing effect between the adsorbed cancer-related substance and silver particles is obtained.
- plasmonic chip of the present invention it is possible to determine the presence or absence of cancer-related substances and chemical modifiers that control chromatin remodeling in biological samples including blood by image diagnosis and Raman analysis.
- a plasmonic chip having a composite needle-like nanocrystal group of silver halide or silver oxide containing halogen, that is, a mesocrystal region of silver oxide containing silver peroxide (FIGS. 9 and 10) is prepared. Serum or a biological sample solution is dropped onto the needle-shaped nanocrystal group region of the monic chip, and a cancer-related substance having a positive charge in the sample is selectively adsorbed.
- FIGS. 1 (a), (b) and (c) Crystal images of cancer-related substances in the case of benign disease, stomach cancer, and colon cancer are shown (FIGS. 1 (a), (b) and (c)). Information such as the area, volume, length, width, etc. of the crystal of this image can be digitized and the numerical value can be made into a histogram.
- a laser microscope Keyence VK-X250 shape analysis laser microscope 15,000 times
- FIGS. 2A, 2B, and 2C there is a crystal between benign disease, stomach cancer, and colon cancer.
- FIG. 4 is an enlarged view of a Raman spectrum when the stomach, as shown in this determination by the peak appearing in the region where the chemical modification of histone tails is said to 1300 cm -1 or less 625 cm -1 or more fingerprint areas of the Raman spectrum It has been shown that it can.
- Cancer-related substances in serum include DNA wound around histones derived from cancer cells, histones (nucleosomes) wound with a single turn of DNA, and chromatin (fibers) in a structure in which they are gathered into a string.
- Globulin is positively charged, but its increase is up to 2 times less than other cancer-related substances, so the substance detected by the present invention increases more than 100 times as cancer progresses. It shows that an increase (cancer-related substance) other than is detected.
- DNA from normal cells, DNA acetylated and dissociated from histones, and albumin occupy about 60% of serum, but are negatively charged and are not adsorbed in the present invention. Therefore, it is convenient for diagnostic imaging of cancer-related substances, and it is convenient for analyzing chemical modification of histone tails by Raman spectroscopy.
- the acicular nanocrystals (silver oxide mesocrystals containing silver peroxide) of the present invention are likely to be negatively charged in an aqueous solution because the silver oxide containing silver peroxide is in contact with the sample (target molecule). It seems to form a charge transfer complex. Furthermore, since silver oxide is reduced by receiving light energy and deposits metallic silver, it has a localized surface plasmon resonance enhancing effect of regularly arranged metal nanoparticles. Therefore, the needle-shaped nanocrystal (mesocrystal) of the present invention is a non-metal, but has both metal properties and ionization properties, so that it becomes a plasmonic chip suitable for surface enhanced Raman scattering (SERS) measurement, and as a cancer diagnostic chip. Convenient.
- SERS surface enhanced Raman scattering
- Quantum crystals formed on metal substrates or metal particles are likely to have positive polarity in aqueous solution as metal complex crystals, and in order to adsorb and fix proteins in biological samples, they are alkaline in the presence of halogen ions. It is preferable to adjust the polarity by dropping a sodium hypochlorite aqueous solution having a pH of 11 or more. Quantum crystals are not only recrystallized and become negative in aqueous solution, but silver oxide composite needle-shaped nanocrystals form peroxides. Can be promoted.
- a nucleosome is a basic structural unit of chromatin, and has a structure in which DNA is wound around a histone octamer composed of four types of histones (H2A, H2B, H3, and H4), and histone plays a role in packaging DNA. In addition, it plays an important role in regulating DNA accessibility and gene regulation. Histone post-translational modification controls interactions with DNA and other nuclear proteins, affecting reversible gene expression. As types of histone modifications, methylation, acetylation, phosphorylation, ubiquitination, SUMOylation, citrullination, and ADP liposylation are mainly known.
- the surrounding gene expression is activated or suppressed.
- histone post-translational modification sites The combination of such histone post-translational modification sites and the effect on gene expression are shown in FIG.
- a histone code-related antibody Genexex anti-histone antibody
- the modified state of the histone code captured by the plasmonic chip of the present invention is observed with a fluorescence image and the histone code hypothesis is verified by Raman spectroscopy. It can be performed.
- the histone code hypothesis is explained as follows.
- chromatin remodeling is a molecular mechanism that regulates gene expression levels through changes in chromatin structure, and with induction, 1) changes in histone modifications, 2) changes in the methylation status of genomic DNA, and 3) This refers to a phenomenon in which a change in the highly sensitive region for DNase occurs.
- the present inventors selectively captured histones and related nucleosomes and chromatin as cancer-related substances as a result of intensive studies. I tried to verify this.
- chromatin remodeling controls nuclear phenomena.
- transcriptional activity varies in a range of about 2500 times depending on the chromatin structure. Since the activation range of transcription factors in naked DNA is about 10 times, most of transcriptional control is performed by chromatin remodeling.
- histone modifiers are chemistry specific to the amino-terminal tail of histones H3 and H4 by acetylation, phosphorylation, methylation, ubiquitination and their removal. A modification pattern is formed. It is the “histone code hypothesis” that when this acts as a recognition code, factors involved in remodeling are further recruited.
- Structural biology is said to play a major role in verifying the "histone code hypothesis" that histone tails that have undergone this chemical modification act as recognition codes for remodeling factors.
- many of HAT, ATP-dependent remodeling factor, coactivator, TFD subunit TAF, etc. have a low homology bromodomain of about 110 amino acids.
- the structure of the bromodomain of coactivator P / CAF has been determined by NMR and a characteristic hydrophobic pocket has been found. Binding experiments using NMR showed that this pocket binds to an acetylated histone H3 tail peptide.
- the crystal structure of two bromodomains arranged in tandem with TAF 250, the largest subunit of the basic transcription factor TFD has also been obtained.
- the transcriptional repressor Mad which has bHLH (basic helix-loophelix) -Zip as a DNA binding domain, recruits a Sin3 complex containing histone deacetylase as a subunit to chromatin.
- the complex of the PAH2 domain of Sin3 and the transcriptional repression domain of Mad has been analyzed by NMR, and the recruitment mechanism to the chromatin site of the deacetylated complex has been discussed.
- genomic DNA which is the main body of genetic material, is very long. In humans, DNA as long as 2 meters is housed in a minute space of only 100 femtoliters, called the cell nucleus. Such storage of genomic DNA is accomplished by a molecular complex called “chromatin”, and the basic structure of chromatin is a “nucleosome” in which DNA is wrapped around histone protein, and the nucleosome is connected in a rosary shape. It binds to proteins and RNAs and is further highly folded to form "higher chromatin”.
- chromatin molecular complex
- chromatin is inhibitory to the functional expression of DNA such as replication, transcription, and recombination.
- organisms can easily replicate, transcribe, and recombine through dynamic changes in chromatin.
- This "dynamic chromatin structure" is created by various nucleosome structures by histone variants and modifications, diversity of their arrangement, interaction with proteins and RNA molecular complexes, etc. It is said to be controlled by the interaction with the nuclear pore complex.
- eukaryotic DNA forms a nucleosome structure that is tightly bound to a protein called histone synthesized in the body, and the nucleosome further spirals to form a chromatin structure.
- acetylated amino group becomes less basic and weakly binds to DNA, and as a result, dissociation of histone and DNA proceeds, and further, acetylation of histone proceeds in the surrounding nucleosomes, and eventually It is said that the promoter is exposed and RNA polymerase is likely to bind. Therefore, in order to elucidate the mechanism of chromatin remodeling based on the histone code hypothesis, how can these cancer-related substances be trapped and analyzed from body fluids containing cancer-related substances such as histones and chromatin? Is important.
- histones and chromatin are trapped as cancer-related substances, and the presence or absence of a cancer symptom can be determined by fluorescence image diagnosis based on the amount of crystals. Then, it can be determined by analyzing the chemical modification of the histone tail and the remodeling factor, which organ cancer the cancer symptom is and the progress of the cancer. And information on how cancers develop and progress through this chromatin remodeling event gives clinicians how they are likely to respond to specific chemotherapeutic agents. In this way, it is possible to rationally design chemotherapy based on knowledge of tumor chemosensitivity, allowing for more accurate predictions.
- the target cancer-related substance to be detected is a protein called histone in which DNA is wound, and this unit structure is called a nucleosome, and the structure in which nucleosomes are gathered and formed into a string is called chromatin (fiber).
- chromatin fiber
- the cells become cancerous and repeat division, tightly wrap around the histone so that the inconvenient gene (tumor suppressor gene) does not come out to increase the number of cancers.
- methylation that makes DNA difficult to unravel easily, but usually histones are charged (+), DNA is charged (-), and two are like magnets They are stuck together and cannot be dissolved by methylation, and the DNA wound around histone is charged to (+) (see FIG. 8 (a)).
- cancer-related substances in blood and biological samples can be selectively detected, and this can be detected by autofluorescence due to the localized plasmon enhancement effect.
- cancer-related substances with various fluorescent labels can be selectively captured and the presence or absence of cancer can be easily determined with a fluorescence microscope.
- the chemical modification state of the histone tail can be determined with or without labeling from the Raman spectrum of the crystal, it is possible to make early determination of cancer and determination regarding the degree of progression of cancer. Therefore, the utilization method of the present invention is summarized as follows. 1. Plasmonic chip using silver quantum crystals No autofluorescence occurs due to antibody dropping and antigen dropping. Therefore, it is possible to observe fluorescence by attaching a fluorescent dye or fluorescent protein.
- Plasmonic chip using silver mesocrystals 1) When a fluorescent substance is attached to the antibody side, the antibody (with label) is solid-phased on the mesocrystal, and an antigen is dropped to measure fluorescence due to binding. 2) When a fluorescent substance is attached to the antigen side, the antibody is solid-phased on the mesocrystal, and the antigen (with a label) is dropped to measure fluorescence due to binding. 3) Measure by autofluorescence. In this case, a fluorescent label can be attached. 3. The crystal on the plasmonic chip is confirmed, the laser is irradiated, and the Raman spectroscopic analysis is performed by the Raman scattering spectrum.
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Abstract
Description
じるといわれ、必要とされない遺伝子をオフにすることにより、DNAメチル化は、生物の
正常な発達および機能にとって不可欠な制御機構である。または、異常なDNAメチル化は
、加齢および多くのがんの発生とともに認められる変化の基礎となる機構の一つでもあるといわれる。
の、メチル基(m)がDNAの特定のシトシン(C)に付加されるDNAの化学的修飾である。この非突然変異(後成的)プロセス(mC)は、遺伝子発現調節における決定的要因であるといわれた(非特許文献1)。そこで、DNAメチル化に注目し、これを検出する観点から結腸がんの早期検出方法が提案されている(特許文献1)。しかし、メチル化DNAを検出することは複雑かつ困難で、時間とコストを要するので簡易迅速検出に不向きである。
式(I):E゜=(RT/|Z|F)ln(βi)
(ここでE゜は、標準電極電位、Rは、気体定数、Tは、絶対温度、Zは、イオン価、Fは、ファラデー定数を表す。)
ここで、金属錯体が、Au、Ag、PtまたはPdから選ばれるプラズモン金属の錯体である場合は、ラマン光に対して局在表面プラズモン共鳴増強効果を有する。特に、金属錯体が銀錯体であるときは、安定度定数(生成定数)(logβi)が8以上の銀錯化剤
とハロゲン化銀との反応により形成されるのがよく、ハロゲン化銀としては塩化銀が好ましく、錯化剤としてはチオ硫酸塩、チオシアン酸塩、亜硫酸塩、チオ尿素、ヨウ化カリ、チオサリチル酸塩、チオシアヌル酸塩から選ばれる1種であるのが好ましい。銀錯体は平均直径が5~20nmであるナノクラスタからなる量子ドットを有し、量子結晶のサイズが100~200nmとなる。
Na2S2O3+4NaClO+H2O →Na2SO4+H2SO4 (2NaHSO4)+4NaCl
Ag+ + NaCl→ AgCl + Na+
Ag+ +3NaOCl → 2AgCl + NaClO3 + 2Na+
Ag+ + OH- → AgOH
2Ag++ 2OH- → Ag2O+H2O
吸着したがん関連物質に紫外、赤色、緑色レーザを照射して蛍光顕微鏡(倍率50倍)で観測した。胃がん(図1(a),(b)及び(c))、大腸がん(図2(a),(b)及び(c))、の場合のがん関連物質の結晶の蛍光画像を示し、良性疾患については蛍光を示さなかった。この画像の結晶の形状、輝度、面積等の情報を数値化し、数値をヒストグラムにすることができる。蛍光顕微鏡で観測すると、良性疾患と胃がん、大腸がんとの間には結晶の形態に差異が見られる。したがって、過酸化銀を含むメソ結晶のプラズモニックチップを用いると、血液中のがん関連物質を選択的に捕捉してがんの存否を蛍光観察して識別することができることがわかる。
血清中のがん関連物質としては、DNAがひと巻きされたヒストン(ヌクレオソーム)、それらが集まりひも状になった構造のクロマチン(線維)を含むと考えられる。グロブリンは正電荷を帯びるが、その増加は他のがん関連物質に比べて最大2倍以下であるので、本発明で検知される物質はがん進行に伴う増加が100倍以上に達するのでグロブリン以外の増加(がん関連物質)が検知されていることを物語っている。また、正常細胞から出るDNA、アセチル化してヒストンから解離したDNA、そしてアルブミンは血清中の約60%を占めるが、負荷電を帯びるため、本発明では吸着されない。したがって、がん関連物質の蛍光画像診断には好都合である。これを確認するため、以下の試験を行った。図15は良性疾患(Benign disease), 胃がん(Gastric cancer),大腸がん( Colon cancer)の 10倍希釈、100倍希釈した場合の100倍、3000倍、3D画像を示す。図16はKatoIII(胃印環細胞がん)培養し、そのがん細胞から抽出した二重鎖DNA、RNA及びプロテンを希釈して観測したものの、(a,b,c)はレーザ顕微鏡を使用したKatoIII(胃印環細胞がん)の、(d,e,f)はそこから抽出したDNAの,(g,h,i)はRNAの、(j,k,l)はプロテンの表面構造を示す100倍、3000倍(画像及びアンジュレーション)、3D画像を示し、(m,n,o)はがん細胞を物理的に粉砕した時の表面構造を示す100倍、1000倍(画像及びアンジュレーション)、3D画像を示す。図17は化学的培養したがん細胞(a),そこから抽出したDNA(b)の,RNA(c)の、プロテン(e)の、物理的に粉砕したがん細胞(e)の、胃がん患者からの血清(f)の、大腸がん患者からの血清(g)の、良性疾患の患者からの血清(h)のメソ結晶プロテオニックチップ上でのラマン散乱スペクトルを示す。なお、KatoIII(胃印環細胞がん)、MKN-45(低分化型胃線がん)、CW-2(日本人由例大腸がん細胞株)、PK45-P(ヒト膵臓がん細胞株)、NHDF-Neo(皮膚線維芽細胞)を示す。これらの結果から本発明のプロテオニックチップによりがん関連物質が選択的に捕捉され、検出されたことを示すものと考えられる。
(実施例1)
チオ硫酸銀1000ppm水溶液を調製し、その1滴をりん青銅板上に滴下し、約3分間放置し、溶液を吹き飛ばすと、SEM像でみると、量子結晶が作成されていた。図は実施例
1で製造したナノ粒子凝集体(量子結晶)の各種SEM像を示す写真であり、100nm前後の薄い六角柱状結晶であって、表面に数nmオーダの凹凸が発現している。金属ナノ結晶に特有のファセットは確認できなかった。図6はりん青銅坂上に滴下後の放置時間と量子結晶形状の関係を示す写真である。まず、六角形の量子結晶が生成し、形状を維持しつつ成長するのが認められる。図4は量子結晶のEDSスペクトル(元素分析)の結果を示すグラフである。りん青銅板上に形成された結晶は銀及び錯体配位子由来の元素を検出したが、銅板上にチオ硫酸銀1000ppm水溶液を調製し、その1滴を滴下し、約3分間放置し、溶液を吹き飛ばした場合は、銀のみを検出したに過ぎなかった。
量子結晶は1000ppmチオ硫酸銀錯体水溶液の場合、りん青銅板上に滴下して3分間放置すると、100nm前後の六角柱状に形成され、各六角柱状の量子結晶は数nmオーダの凹凸を持つことがSEM像から確認されたが、金属ナノ結晶に特有のファセットは確認できず、EDS元素分析で銀及び錯体配位子由来の元素を検出されたため、全体は銀錯体のナノ結晶であって、その表面に現れる凹凸は錯体中の銀がクラスタとして量子ドットを形成して広がっていると推測される。本発明の銀錯体量子結晶がりん青銅板上に形成される一方、銅基板上には銀のみのナノ粒子が析出する現象を見ると、チオ硫酸銀錯体の平衡電位が0.33で銅の電極電位(0.34)と同等であるため、銅基板上には銀(0.80)のみが析出し、りん青銅の場合は0.22と電極電位がわずかに卑であるため、銀錯体の結晶が析出したものと思われる。したがって、量子結晶を作成するためには1)錯体水溶液が500~2000ppmという希薄な領域であること、2)金属錯体水溶液の平衡電位に対し担持金属の電極電位がわずかに卑であること、3)電極電位差で金属錯体が凝集させることが重要であると思われる。また、1000ppmチオ尿素銀錯体水溶液を使用した場合も同様であった。
上記量子結晶基板に5%次亜塩素酸ソーダ水溶液を滴下して2分間処理して除去すると図11に示す結晶構造が見られ、針状の結晶とラクビーボール状の塊と大きい塊が見られたので、それぞれの組成をEDSスペクトル(元素分析)で分析すると、以下の反応式から
針状の結晶はともに塩化銀と酸化銀の複合結晶からなるものと考えられるが、図7の結果は塩素は確認できず、銀と酸素が支配的であることがわかる。
Na2S2O3+4NaClO+H2O →Na2SO4+H2SO4+4NaCl (1)
Ag+ + NaCl→ AgCl + Na+ (2)
Ag+ +3NaOCl → 2AgCl + NaClO3 + 2Na+ (3)
Ag+ + OH- → AgOH (4)
2Ag+ + 2OH- → Ag2O +H2O (5)
したがって、本発明に係るメソ結晶の形成には銀イオンとチオ硫酸イオンが塩素イオンの存在下にアルカリ酸化反応により生ずるものと思われるが、通常の水溶液中では酸化銀が形成されるに過ぎないが、以下のXPS測定から過酸化銀が支配的に形成されていると推測される。
XPS測定:
上記量子結晶基板に次亜塩素酸ナトリウム水溶液25μlを2分間滴下し、再結晶基板を作り、エッチングせずそのまま(使用機種:アルバック・ファイ(株)/PHI5000 Versa Probe II(走査型X線光電子分光分析装置))でAgとOとをXPS測定した。また、比較対象
のため、酸化銀の粉と塩化銀の粉のAgを測定した。他方、再結晶基板をアルゴンガスクラスターイオン銃で5分間エッチングしてAgとOをXPS測定した。図9及び図10のXPS測定結果を図8に基づくEDSの結果から推測すると、529eV付近のピークは過酸化銀(AgO)に由来するOピークで、530eV付近のピークは酸化銀(Ag2O)に由来するO
ピークであると認められる。エッチングした場合に、酸素量は減少するが、529eV付近のピークの過酸化銀(AgO)に由来するOピークが、530eV付近のピークは酸化銀(Ag2O)に由来するOピークよりも大きいことは基板近傍に過酸化銀が形成されているのを物語るものといえる。これは、メソ結晶形成時の触媒作用と基板の電極電位が影響しているものと推測される。
なお、EDS測定は上記再結晶基板を使用機種:日本電子株式会社/JSM-7001F(電界
放出形分析走査電子顕微鏡)を用いて行った。
また、チオ硫酸銀の量子結晶を次亜塩素酸水溶液、0.01規定苛性ソーダ水溶液、0.01規定塩酸水溶液、0.1モル炭酸ナトリウム水溶液で処理しても同様の結果は得られなかった。よって、この針状結晶の形成には銀イオンとチオ硫酸イオンの存在下に上記反応により生ずるものと思われる。酸化銀は水溶液中で負電荷を帯び、光により還元されて金属銀を析出させる。過酸化銀はその傾向が顕著なので、正電荷のがん関連物質を吸着し、しかも吸着したがん関連物質と銀粒子との間の表面プラズモン増強効果が得られるものと思われる。
吸着したがん関連物質を反射顕微鏡(倍率500倍)で観測した。良性疾患、胃がん、大腸がんの場合のがん関連物質の結晶画像を示す(図1(a),(b)及び(c))。この画像の結晶の面積、体積、長さ、幅等の情報を数値化し、数値をヒストグラムにすることができる。レーザー顕微鏡(キーエンス社製VK-X250形状解析レーザ顕微鏡 1万5千倍)で観測すると、図2A,図2B,図2Cに示すように、良性疾患と胃がん、大腸がんとの間には結晶の形態に差異が見られる。この画像のR,G、Bをヒストグラムでグラフ化すると、図2A,図2B,図2Cのグラフに示すように、良性疾患と胃がん、大腸がんとの間には明らかにピークの位置および形状に差異が認められる。したがって、過酸化銀を含むメソ結晶のプラズモニックチップを用いると、血液中のがん関連物質を選択的に捕捉してがんの存否を識別することができることがわかる。なお、共焦点レーザー顕微鏡を用いると、スライス画像が得られるため、内部の情報が得られるので、好ましい。
また、良性疾患(食道アカラシア、胃間葉系腫瘍、十二指腸良性腫瘍)の患者から得られた血清とがん患者(胃がん)、大腸がん,膵がん)から得られた血清のプラズモニックチップ上の自家蛍光を蛍光顕微鏡で観察すると、図20に示されるように良性疾患とがん患者との間には自家蛍光が観測されるか否かで明確な識別ができることがわかる。
プラズモニックチップ上に吸着したがん関連物質の結晶は目視では観測できないが、顕微鏡(500倍)で観測すると、点状に分散した結晶塊が観測される(図18)。この結晶塊の一つに対しレーザー(波長532nm)照射してそこからのラマンスペクトルを検知することにより、表面増強ラマン散乱(SERS)の強度によりがん疾病を判断することができる。図3(a),(b),(c)は良性疾患、胃がん、及び大腸がんの場合のプラズモニックチップ上の結晶に対しレーザー(波長532nm)照射してそこから得られたラマンスペクトルを示す。特に、図4は胃がんの場合のラマンスペクトルの拡大図で、これに示すように、ヒストンテールの化学修飾はラマンスペクトルの1300cm-1以下625cm-1以上の指紋領域といわれる領域に現われるピークによって判別できることが示されている。
ヌクレオソームはクロマチンの基本的構成単位で、4種のヒストン(H2A、H2B,H3、H4)からなるヒストン8量体にDNAが巻きついた構造をしているが、ヒストンはDNAをパケージングするという役割に加え、DNAのアクセシビリティを調節すること及び、遺伝子制御にも重要な役割を果たしている。ヒストンの翻訳後修飾により、DNAやその他の核蛋白質との相互作用が制御され、可逆的な遺伝子発現に影響を及ぼす。ヒストン修飾の種類として、メチル化、アセチル化、リン酸化、ユビキチン化、SUMO化、シトルリン化、ADPリポシル化、が主として知られている。ヒストンの配列中、どの部位がこれらの修飾を受けるかによって、周囲の遺伝子発現は活性化または抑制されるが、こうしたヒストンの翻訳後修飾部位の組み合わせと遺伝子発現への影響を図18に示す各種ヒストンコード関連抗体(Genetex社抗ヒストン抗体)を使用すると、本発明のプラズモニックチップで捕捉されたヒストンコードの修飾された状態を、蛍光画像で観測するとともにラマン分光分析により、ヒストンコード仮説の検証を行うことができる。
なお、ヒストンコード仮説では次のように説明されている。
ロックフェラー大学のアリス博士らのグループによりヒストンコード仮説がクロマチンリモデリングとの関係で提唱されている。即ち、クロマチンリモデリングとはクロマチン構造の変化を介して遺伝子の発現レベルを調節する分子機構、誘導に伴い,1)ヒストン修飾の変化,2)ゲノムDNAのメチル化状態の変化,それに伴う3)DNaseに対する高感受性領域の変化が起こる現象をいう。本発明者らは、ヒストンコード仮説に基づく、がん発生の機序を解明すべく、鋭意研究の結果、がん関連物質として、ヒストン及びこれと関連するヌクレオソーム並びにクロマチンを選択的に捕捉し、これを検証しようとした。ゲノムDNAのほとんどはコアヒストンに巻き取られ、クロマチンとして格納されている。そのためゲノムDNA に直接働きかける転写、複製、修復などの核内現象は何らかの形でのクロマチン構造変化を伴う。この広義のクロマチンリモデリングは逆に核内現象を制御している。例えば転写活性はクロマチン構造に依存して約2500倍の範囲で変化する。裸のDNAにおける転写因子の活性化のレンジは10倍程度なので、転写制御の大半はクロマチンリモデリングで行われていることになる。このクロマチンリモデリングに関与する因子の中で、ヒストン修飾因子はアセチル化、リン酸化、メチル化、ユビキチン化やそれらの除去を行うことによって、ヒストンH3、H4のアミノ末端側のテールに特有な化学修飾パターンを形成する。これが認識コードとして働くことによってさらにリモデリングに関与する因子がリクルートされるというのが"ヒストンコード仮説"である。
クチベーターP/CAF のブロモドメインの構造がNMR により決定され、特徴的な疎水性ポケットが見つかっている。NMRを使った結合実験の結果、このポケットはアセチル化されたヒストンH3テールのペプチドと結合することが示された。また基本転写因子TFDの最大サブユニットであるTAF 250 のタンデムに並んだ2つのブロモドメインの結晶構造も得られている。これはアセチル化を受けたヒストンH4のテールに結合する活性を持つ。またheterochromatin protein 1のクロモドメインがメチル化されたヒストンH3テールに結合することが示されている。マウスchromatinmodifier protein 1 のクロモドメインのNMR構造が報告されている。このクロマチンリモデリング因子はヒストンテールのコードをブロモドメインなどが認識して直接的に特定のヌクレオソーム部位にアクセスすることもあるが、DNA 結合性の転写因子によって間接的にリクルートされる場合も多い。bHLH(basic helix-loophelix)-ZipをDNA結合ドメインとして持つ転写リプレッサーMad は、ヒストン脱アセチル化酵素をサブユニットとして含むSin3複合体をクロマチンにリクルートする。Sin3のPAH2ドメインとMad の転写抑制ドメインの複合体がNMRによって解析され、脱アセチル化複合体のクロマチン部位へのリクルート機構が議論されている。
やDNA変異率の低減が見られる。またゲノムDNAのメチル化パターンとゲノムインプリンティング、X 染色体不活性化や細胞の腫瘍化には明確な相関が見られるからである(非特許文献2)。したがって、ヒストンおよびクロマチンの構造解析はがんとの関連を説く鍵を握っており、ヒストンテールを化学修飾する因子を解析することは重要な意義を持っているといえる。本発明ではヒストンおよびクロマチンを選択的に捕捉することができるので、ヒストンコードの化学修飾状態を蛍光標識化するとともに、表面増強ラマン分析(SERS)を利用して解析する手段を提供する。
ところで、遺伝物質の本体であるゲノムDNAは長大で、ヒトでは長さ2メートルものDNAが、細胞核というわずか体積100フェムトリットルの微小空間に収納されている。このよ
うなゲノムDNAの収納は、"クロマチン"と呼ばれる分子複合体によって成し遂げられ、ク
ロマチンの基盤構造は、ヒストンタンパク質にDNAが巻き付いた"ヌクレオソーム"で、そ
のヌクレオソームが数珠状につながったものが、タンパク質やRNAと結合してさらに高度
に折り畳まれ"高次のクロマチン"を形成しているというものである。しかし、クロマチンからDNAがほどけるためには、大きなエネルギーが必要で、クロマチンは、複製、転写、組換えなどのDNAの機能発現に阻害的である。しかし生物は、クロマチンの動的変動を介
して、いとも簡単に複製、転写、組換えをやってのける。この"動的クロマチン構造"は、ヒストンバリアントや修飾による多様なヌクレオソーム構造、その並び方の多様性、タンパク質やRNA分子複合体との相互作用などによって生み出されて、細胞核構造体、核膜、
核膜孔複合体などとの相互作用によって制御されているといわれる。上記現象を説明すると、真核生物のDNAは、体内で合成されたヒストンというタンパク質と強固に結合した、ヌクレオソーム構造を形成し、ヌクレオソームは、さらにらせんになってクロマチン構造を形成するが、このように、ヒストンとDNAとがしっかり結合した状態では、RNAポリメラーゼがDNAに結合することが難しく、他方、転写が盛んになるときは、クロマチン構造が緩んで、ヌクレオソームからヒストンが外れてDNAが裸に近くなる。転写が抑制される時には、逆に、ヌクレオソーム構造がしっかりして、クロマチンが凝集する。このようなクロマチンの構造の再構築や再構成を上述したクロマチンリモデリングというが、プロモーターやエンハンサーは転写を促進し、遺伝子発現を高める。これらの役割の一つは、ヒストンとDNAの結合を緩め、時にはヌクレオソーム構造を破壊して、RNAポリメラーゼによるRNA合成を促進する。ヌクレオソームをもう少し詳しく見ると、翻訳済みのヒストンタンパク質の末端にあるヒストンテイルの部分に、塩基性アミノ酸(プラス電荷)がたくさん存在する。これが、DNAのリン酸基(マイナス電荷)と静電的結合をする。ヒストンとDNAの結合を緩めるには、ヒストンの強い塩基性を緩めればよい。クロマチンリモデリングによる転写調節は、実際には多くの種類の酵素やタンパク質が関わる複雑な反応で、プロモーターやエンハンサーに結合する転写因子には、ヒストンアセチル化酵素(HAT:histone acetyltransferase)が結合して、この酵素がヒストンのアミノ基をアセチル化する。アセチル化したアミノ基は、塩基性が低下してDNAとの結合が弱くなり、これを契機にしてヒストンとDNAの解離が進行し、さらに周囲のヌクレオソームでヒストンのアセチル化が進行して、やがてプロモーターが露出して、RNAポリメラーゼが結合しやすくなるといわれる。したがって、ヒストンコード仮説に基づくクロマチンリモデリングの機序を解明するためにはヒストン、クロマチン等のがん関連物質を含む体液から如何にしてこれらがん関連物質をトラップし、分析することができるかは重要である。
1.銀量子結晶を用いるプラズモニックチップ
抗体滴下及び抗原滴下により自家蛍光はおきない。したがって、蛍光色素、蛍光蛋白をつけることにより蛍光観測が可能となる。エンドトキシンではエンドトキシンとの結合ピークがラマン散乱スペクトルで確認できているため、蛍光標識を付けることによってより明確に識別することができる。よって、1)抗体側に蛍光物質をつける場合、金属錯体と抗体の混合液を本発明のプラズモニックチップに滴下し、抗原が結合すると発光するのを、観測する。他方、2)抗原側に蛍光物質を付ける場合、量子結晶に抗体を滴下して固相化し、抗原側に予め蛍光物質をつけて抗原抗体反応により蛍光が発光するのでこれを測定する。
2.銀メソ結晶を用いるプラズモニックチップ
1)抗体側に蛍光物質をつける場合、メソ結晶に抗体(標識付き)を固相化し、抗原を滴下して結合による蛍光を測定する。2)抗原側に蛍光物質を付ける場合、メソ結晶に抗体を固相化し、抗原(標識付き)を滴下して結合による蛍光を測定する。3)自家蛍光による測定する。この場合に蛍光標識を付けることもできる。
3.プラズモニックチップ上の結晶を確認し、レーザを照射してラマン散乱スペクトルによりラマン分光分析を行う。
Claims (6)
- 蛍光顕微鏡で観測する蛍光画像観察用基板であって、プラズモン金属として銀錯体水溶液をそのプラズモン金属錯体の還元電位近傍の、より卑なる電極電位を有する金属基板上に電極電位差により形成される金属錯体量子結晶領域を有し、銀錯体水溶液中に腫瘍マーカー、抗体等の吸着物質を金属策錯体の量子結晶とともに配位させ、がん連物質、抗原等の被吸着物質を選択的に捕捉し、局在表面プラズモン共鳴機能で増強した蛍光画像観察するためのプラズモニックチップ。
- 蛍光顕微鏡で観測する蛍光画像観察用基板であって、金属基板上に形成される銀錯体量子結晶をアルカリ処理してなる過酸化銀を含む銀酸化物のナノ結晶からなるメソ結晶領域を有し、該銀酸化物のメソ結晶でがん関連物質を捕捉し、メソ結晶領域に光照射による、局在表面プラズモン共鳴機能で増強したがん関連物質の自家蛍光による画像観察するためのプラズモニックチップ。
- 請求項1又は2記載のプラズモンチップを用意し、該プラズモニックチップの結晶領域に血清又は生体試料液を滴下し、試料中のがん関連物質、抗原等の吸着物質を選択的に吸着し、プラズモニックチップ上に凝集する結晶領域に光照射し、局在表面プラズモン共鳴によってがん関連物質の自家蛍光又はがんマーカーを蛍光顕微鏡で蛍光画像診断してがんの有無を診断することを特徴とするがん疾病の蛍光画像診断方法。
- 請求項1又は2記載のプラズモンチップを用意し、該プラズモニックチップの結晶領域に血清又は生体試料液を滴下し、試料中のがん関連物質、抗原等の吸着物質を選択的に吸着し、プラズモニックチップ上に凝集する結晶領域に光照射し、局在表面プラズモン共鳴によってがん関連物質の自家蛍光又はがんマーカーを蛍光顕微鏡で蛍光画像診断してがんの有無を診断した後、自家蛍光又はがんマーカーの蛍光画像を目印にレーザー光を照射してラマン分光分析することを特徴とするがん疾病のラマン分光診断方法。
- がん関連物質がDNAが巻きついたヒストンを含むヌクレオソーム又はクロマチンである請求項3記載のがん疾病の蛍光画像診断方法。
- がん関連物質がDNAが巻きついたヒストンを含むヌクレオソーム又はクロマチンである請求項4記載のがん疾病のラマン分光診断方法。
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KR20220136354A (ko) | 2019-12-25 | 2022-10-07 | 유겐가이샤 마이테크 | 아포토시스에 의하여 세분화된 뉴클레오솜을 표적으로 하는 자가 형광에 의한 리퀴드·바이옵시법 |
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KR102232197B1 (ko) | 2021-03-25 |
JP6492066B2 (ja) | 2019-03-27 |
SG11201609331XA (en) | 2016-12-29 |
ES2905092T3 (es) | 2022-04-07 |
US10215700B2 (en) | 2019-02-26 |
IL248760B (en) | 2021-01-31 |
JP2016208959A (ja) | 2016-12-15 |
IL248760A0 (en) | 2017-01-31 |
CN106461556A (zh) | 2017-02-22 |
MY193573A (en) | 2022-10-19 |
NZ726748A (en) | 2020-05-29 |
EP3141888A1 (en) | 2017-03-15 |
CA2948353A1 (en) | 2015-11-12 |
HUE057218T2 (hu) | 2022-04-28 |
CN106461556B (zh) | 2019-12-27 |
EP3141888A4 (en) | 2018-03-14 |
CA2948353C (en) | 2021-12-14 |
AU2015256932B2 (en) | 2019-11-07 |
JPWO2015170711A1 (ja) | 2017-04-20 |
KR20170007774A (ko) | 2017-01-20 |
EP3141888B1 (en) | 2021-11-03 |
AU2015256932A1 (en) | 2016-12-08 |
US20170115216A1 (en) | 2017-04-27 |
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