The present invention relates to a system capable of performing simple and rapid inspection of an antigen equivalent to the immune chromatographic method with accuracy good as a PCR method. An embodiment relates to a novel fluorescence counting system for quantifying viruses or antibodies in an analyte which comprises an unit of providing an antigen or antibody phase solidified substrate by an aggregation method with quantum crystals, an unit for making a labeling liquor and labeling a virus or an antibody to be measured in the analyte by an antigen-antibody method, an unit of exciting the fluorescently labeled virus or antibody by a surface plasmon excitation method, and an unit of counting fluorescent points in an excited fluorescent screen to quantify the virus or antibody in the analyte.
[Problem] Provided is a method for detecting of an autofluorescence Liquid Biopsy of Methylated Fragmented DNA (fragmented nucleosome) released into the blood by cell apoptosis as a disease-related substance
[Problem] Provided is a method for detecting of an autofluorescence Liquid Biopsy of Methylated Fragmented DNA (fragmented nucleosome) released into the blood by cell apoptosis as a disease-related substance
[Solution] The inventive method comprises a) a step of capturing the fragmented DNA (fragmented nucleosome) in the analyte as a disease-related substance onto the plasmonic metal meso-crystals; b) a step of irradiating the captured fragmented DNA (fragmented nucleosome) on the plasmonic metal meso-crystal with excitation light to enhance the autofluorescence by the surface plasmon enhancing effect, and acquiring a fluorescent colony image via a filter in a longer wavelength range than the excitation light filter; c) a step of adopting a pixel that exhibits a brightness greater than or equal to a predetermined threshold value of said fluorescent colony image; d) calculating a ratio of a total area value of pixels greater than or equal to a predetermined threshold value of a different two-wavelength region of the adopted measurement region.
[Problem] To provide a method for: using, as a disease-related substance, fragmented DNA (segmented nucleosome) released in blood as a result of cell apoptosis; and spectroscopically analyzing the autofluorescence of the fragmented DNA to easily and rapidly detect a disease. [Solution] The present invention is a method for spectroscopically analyzing autofluorescence, the method being characterized by including a step for averaging the dispersion spectra of a plurality of fluorescent points, and a step for: allowing plasmon metal mesocrystal to charge-trap, as a disease-related substance, fragmented DNA (segmented nucleosome) present in a sample; irradiating with a laser beam of a short wavelength to intensify, using the surface plasmon intensification effect, the autofluorescence of the fragmented DNA; determining a certain region of interest (ROI) in the fluorescence image of the fluorescence colony of the fragmented DNA (segmented nucleosome); and dispersing, with the liquid biopsy method for observing the autofluorescence of the region of interest that has been determined, a fluorescent point that is in the region of interest which has been determined and that is equal to or above a prescribed threshold, within a wavelength width of 0.5-30 nm to acquire a dispersion spectrum. The method is also characterized by detecting the peak top wavelength of the averaged spectrum.
The present invention relates to a system capable of performing simple and rapid inspection of an antigen equivalent to the immune chromatographic method with accuracy good as a PCR method. An embodiment relates to a novel fluorescence counting system for quantifying viruses or antibodies in an analyte which comprises an unit of providing an antigen or antibody phase solidified substrate by an aggregation method with quantum crystals, an unit for making a labeling liquor and labeling a virus or an antibody to be measured in the analyte by an antigen-antibody method, an unit of exciting the fluorescently labeled virus or antibody by a surface plasmon excitation method, and an unit of counting fluorescent points in an excited fluorescent screen to quantify the virus or antibody in the analyte.
G01N 21/78 - Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator producing a change of colour
G01N 33/543 - ImmunoassayBiospecific binding assayMaterials therefor with an insoluble carrier for immobilising immunochemicals
G01N 33/569 - ImmunoassayBiospecific binding assayMaterials therefor for microorganisms, e.g. protozoa, bacteria, viruses
5.
Fluorescence counting system for quantifying viruses or antibodies on an immobilized metal substrate by using an antigen-antibody reaction
The present invention relates to a system capable of performing simple and rapid inspection of an antigen equivalent to the immune chromatographic method with accuracy good as a PCR method. An embodiment relates to a novel fluorescence counting system for quantifying viruses or antibodies in an analyte which comprises an unit of providing an antigen or antibody phase solidified substrate by an aggregation method with quantum crystals, an unit for making a labeling liquor and labeling a virus or an antibody to be measured in the analyte by an antigen-antibody method, an unit of exciting the fluorescently labeled virus or antibody by a surface plasmon excitation method, and an unit of counting fluorescent points in an excited fluorescent screen to quantify the virus or antibody in the analyte.
The present invention relates to a system capable of performing simple and rapid inspection of an antigen equivalent to the immune chromatographic method with accuracy good as a PCR method. An embodiment relates to a novel fluorescence counting system for quantifying viruses or antibodies in an analyte which comprises an unit of providing an antigen or antibody phase solidified substrate by an aggregation method with quantum crystals, an unit for making a labeling liquor and labeling a virus or an antibody to be measured in the analyte by an antigen-antibody method, an unit of exciting the fluorescently labeled virus or antibody by a surface plasmon excitation method, and an unit of counting fluorescent points in an excited fluorescent screen to quantify the virus or antibody in the analyte.
G01N 21/78 - Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator producing a change of colour
[Problem] To provide a solid-phase substrate for detecting quantities of inactivated antigens or antibodies in a sample using surface plasmon-enhanced fluorescence spectroscopy (SPFS). [Solution] The present invention is a solid-phase substrate in which plasmon metal complexes in a solution, through selection of an electrodeposition substrate potential, coagulate as quantum crystals on a metal substrate with an electrode potential near a reduction potential. When this occurs, if antigens, portions thereof, or antibodies are also present in the solution, the antigens, the portions thereof, or the antibodies coagulate on the substrate or on particles together with the metal complexes, completing a solid-phase plasmon reaction field. Further, although being a solid-phase substrate, the substrate, unlike a plasmon metal thin film, has a regular arrangement of hexagonal plate–shaped metal complex crystals around 100 nm in size, and the antigens, the portions thereof, or the antibodies enter solid phase physically or chemically between these quantum crystals at certain intervals. Therefore, as in the formation of microflow paths, the substrate is applicable to surface plasmon enhancement and can be used in a fluorescence detection method for virus antigens, portions thereof, and virus antibodies. A fluorescence image is observed using a fluorescence microscope or a fluorescence reader, and fluorescent particles with a brightness that is at least a given value are binarized within a given scope of the obtained fluorescence image or from the entire image, and the obtained quantity can be detected by counting.
[Problem] Provided is a method for detecting of an autofluorescence Liquid Biopsy of Methylated Fragmented DNA (fragmented nucleosome) released into the blood by cell apoptosis as a disease-related substance[Solution] The inventive method comprises a) a step of capturing the fragmented DNA (fragmented nucleosome) in the analyte as a disease-related substance onto the plasmonic metal meso-crystals ; b) a step of irradiating the captured fragmented DNA (fragmented nucleosome) on the plasmonic metal meso-crystal with excitation light to enhance the autofluorescence by the surface plasmon enhancing effect, and acquiring a fluorescent colony image via a filter in a longer wavelength range than the excitation light filter; c) a step of adopting a pixel that exhibits a brightness greater than or equal to a predetermined threshold value of said fluorescent colony image; d) calculating a ratio of a total area value of pixels greater than or equal to a predetermined threshold value of a different two-wavelength region of the adopted measurement region.
[Problem] To provide a method that targets fragmented DNA (fragmented nucleosomes) released into the blood by cellular apoptosis as a disease-associated substance and makes it possible to quickly and easily detect disease by liquid biopsy using the autofluorescence of methylated fragmented DNA (fragmented nucleosomes) collected from a body fluid such as blood. [Solution] The present invention is a liquid biopsy method based on the autofluorescence of fragmented DNA (fragmented nucleosomes) that includes a) a step for bringing a specimen that comprises a diluted or undiluted body fluid or culture solution that includes cells into contact, within a sample, with a measurement substrate that has a plasmon metal mesocrystal region and charge trapping fragmented DNA (fragmented nucleosomes) in the specimen onto the plasmon metal mesocrystals as a disease-associated substance, b) a step for shining excitation light at the fragmented DNA (the fragmented nucleosomes) trapped on the plasmon metal mesocrystals, amplifying the autofluorescence thereof by surface plasmon amplification, setting a fixed measurement region (ROI) of a fluorescence image of fluorescence colonies of the fragmented DNA (the fragmented nucleosomes), and acquiring a fluorescence colony image via a filter that has a longer wavelength range than an excitation light filter, c) a step for selecting pixels of the fluorescence colony image that have a brightness that is at or above a prescribed threshold value, and d) a step for calculating a total area value for the selected pixels in the measurement region that are at or above a prescribed threshold value in a prescribed wavelength range or a ratio for total area values for the selected pixels in the measurement region that are at or above a prescribed threshold value in two different wavelength ranges.
[Problem] To provide a quick and easy detection method that uses a liquid biopsy method that is based on the autofluorescence of a disease-associated substance. [Solution] The present invention includes a) a step for bringing a specimen that is a culture liquid that includes a body fluid or cells, or a specimen that has been prepared by diluting a culture liquid that includes a body fluid or cells, into contact with a measurement substrate that has a plasmon metal nanocrystal region that exhibits a surface negative charge in a sample and charge trapping protein conjugates in the specimen that exhibit a positive charge on the plasmon metal nanocrystals as a disease-associated substance, b) a step for radiating excitation light at the protein conjugates that have been trapped on the plasmon metal nanocrystals to amplify the autofluorescence of the trapped protein conjugates by surface plasmon amplification and acquiring a fluorescence image of fluorescence colonies, c) a step for binarizing the luminance of the fluorescence colonies and selecting fluorescence colonies that have a luminance that is at or above a prescribed threshold value, and d) a step for computing a ratio value using a total luminance value and/or a total area value for the selected fluorescence colonies that are at or above the prescribed threshold value or using RGB and/or a two-wavelength ratio that corresponds thereto for the selected fluorescence colonies that are at or above the prescribed threshold value.
Object: To provide a biochip for use in exhaustive analysis of a particular protein including DNA (deoxyribose nucleic acid) in a body fluid through Raman quantitative analysis.
(wherein E° represents the standard electrode potential, R represents a gas constant, T represents the absolute temperature, Z represents the ion valency, and F represents the Faraday constant), the surface property of the metal complex quantum crystals on the carrier metal being subsequently adjusted in dependence on an object to be detected in the aqueous solution prior to the precipitation or after the precipitation.
A cancer-related substance in blood or a biological sample can be detected in a selective manner, so it becomes possible to determine the occurrence of cancer by observation of fluorescent image of a crystal of the censor related substance or a coagulated state of the crystal on a plasmonic chip. In addition, the state of chemical modification of a histone tail can be determined by a Raman spectrum analysis. Furthermore, the location of the cancer-related substance aggregated on a substrate cannot be determined with naked eyes. Then, as a second aspect of the present invention, a method for diagnosing a cancer disease is provided, said method being characterized by firstly identifying the location of the region of a crystal by observing fluorescent image on a microscope, and then irradiating the crystal, with laser beam to analyze with respect to the chemical modification of a histone tail and a remodeling factor.
A PLASMONIC CHIP AND A DIAGNOSTIC METHOD FOR OBSERVATION OF A FLUORESCENCE IMAGE AND RAMAN SPECTROSCOPY OF CANCER-RELATED SUBSTANCE ON THE PLASMONIC CHIPS
[Problem] To provide: a plasmonic substrate; a method for the diagnostic fluorescent imaging of a related substance or a tumor marker using the plasmonic substrate; and a Raman spectroscopic analysis method using the plasmonic substrate. [Solution] In the present invention, a cancer-related substance in blood or a biological sample can be detected in a selective manner, and therefore it becomes possible to determine the occurrence of cancer by the diagnostic fluorescent imaging of a crystal or the coagulated state of the crystal on a plasmonic chip. In addition, the state of chemical modification of a histone tail can be determined by a Raman spectrum of the crystal, and therefore it becomes possible to detect the occurrence of cancer at an earlier stage and to determine the degree of progression of cancer. Furthermore, the location of a cancer-related substance aggregated on a substrate cannot be determined with naked eyes. Then, as a second aspect of the present invention, a method for diagnosing a cancer disease is provided, said method being characterized by comprising identifying the location of the region of a crystal by a diagnostic imaging on a microscope, and then irradiating the crystal, of which the location has been identified, with laser beam to analyze with respect to the chemical modification of a histone tail and a remodeling factor.
[Problem] To provide: a plasmonic substrate; a method for the diagnostic fluorescent imaging of a related substance or a tumor marker using the plasmonic substrate; and a Raman spectroscopic analysis method using the plasmonic substrate. [Solution] In the present invention, a cancer-related substance in blood or a biological sample can be detected in a selective manner, and therefore it becomes possible to determine the occurrence of cancer by the diagnostic fluorescent imaging of a crystal or the coagulated state of the crystal on a plasmonic chip. In addition, the state of chemical modification of a histone tail can be determined by a Raman spectrum of the crystal, and therefore it becomes possible to detect the occurrence of cancer at an earlier stage and to determine the degree of progression of cancer. Furthermore, the location of a cancer-related substance aggregated on a substrate cannot be determined with naked eyes. Then, as a second aspect of the present invention, a method for diagnosing a cancer disease is provided, said method being characterized by comprising identifying the location of the region of a crystal by a diagnostic imaging on a microscope, and then irradiating the crystal, of which the location has been identified, with laser beam to analyze with respect to the chemical modification of a histone tail and a remodeling factor.
[Problem] To provide a Raman quantification method for measuring cancer cell-derived free DNA. [Solution] This Raman quantification method is characterized by involving a step for preparing a biochip having a mesocrystal region of a silver oxide containing peroxide silver, adding blood serum or biological sample solution dropwise onto the mesocrystal region of said biochip, selectively adsorbing the cancer-related substance having a positive charge in the sample, irradiating the adsorbed cancer-related substance with a laser and detecting Raman scattering therefrom, wherein the cancer illness is determined on the basis of the intensity of the surface-enhanced Raman spectroscopy (SERS). In the carbon-specific D band and G band in the Raman scattering spectrum, a characteristic peak spectrum of the cancer-related substance can be detected in the proximity of the methyl group-characteristic 2900cm-1.
[Problem] To provide a biochip for comprehensively analyzing specific proteins including DNA in a biological fluid by Raman quantitative analysis. [Solution] A biochip is provided which is formed by adding a metal complex aqueous solution containing a complex of a plasmon metal selected from Au, Ag, Pt and Pd dropwise onto a support metal having an electrode potential lower than that of the complex metal, and precipitating the metal complex on the carrier metal as nanosize quantum crystals. The metal complex is selected so as to have a complex stability constant (logβ) no less than that indicated by formula (I), which correlates with the electrode potential (E) of the support metal. Formula (I): E° = (RT/|Z|F)ln(βi) (Here, E° is the reference electrode potential, R is the gas constant, T is the absolute temperature, Z is the ionic valency, and F is the Faraday constant.) In an aqueous solution before precipitation or after precipitation, the metal complex quantum crystals on the support metal adjust the surface properties of the biochip in accordance with the detection target.
[Problem] To provide a Raman quantification method for measuring cancer cell-derived free DNA. [Solution] This Raman quantification method is characterized by involving a step for preparing a biochip having a mesocrystal region of a silver oxide containing peroxide silver, adding blood serum or biological sample solution dropwise onto the mesocrystal region of said biochip, selectively adsorbing the cancer-related substance having a positive charge in the sample, irradiating the adsorbed cancer-related substance with a laser and detecting Raman scattering therefrom, wherein the cancer illness is determined on the basis of the intensity of the surface-enhanced Raman spectroscopy (SERS). In the carbon-specific D band and G band in the Raman scattering spectrum, a characteristic peak spectrum of the cancer-related substance can be detected in the proximity of the methyl group-characteristic 2900cm-1.
The present quantum crystals are produced by a method characterized in that an aqueous solution of plasmon metal complex made from a ligand and a plasmon metal selected from the group consisting of gold, silver, copper, nickel, zinc, aluminum, and platinum is prepared and brought into contact with a carrier made of a metal or a metal alloy having an electrode potential lower than an electrode potential of the plasmon metal in the aqueous solution. When the plasmon metal complex is precipitated as quantum crystals arranged on the carrier, the metal complex crystals are formed as metal quantum dots.
B82B 3/00 - Manufacture or treatment of nanostructures by manipulation of individual atoms or molecules, or limited collections of atoms or molecules as discrete units
B82Y 15/00 - Nanotechnology for interacting, sensing or actuating, e.g. quantum dots as markers in protein assays or molecular motors
B82Y 30/00 - Nanotechnology for materials or surface science, e.g. nanocomposites
B82Y 40/00 - Manufacture or treatment of nanostructures
C23C 16/50 - Chemical coating by decomposition of gaseous compounds, without leaving reaction products of surface material in the coating, i.e. chemical vapour deposition [CVD] processes characterised by the method of coating using electric discharges
[Problem] To provide a substrate for SPR and SERS analysis of a biochemical substance with the use of surface enhanced Raman scattering and an analysis method using the same. [Solution] A substrate comprising metal complex quantum crystals that have been deposited on a metal substrate from a dilute plasmon metal complex solution, or a receptor solid phase substrate having a receptor such as an antibody that has been immobilized on the aforesaid substrate, said substrate being applicable to an SPR or SERS analysis method. By using the substrate, the presence or content of a biochemical substance contained in a subject to be analyzed is detected with the use of surface enhanced Raman scattering. Thus, the subject to be analyzed binds to the receptor such as an antibody that has been immobilized on the metal complex quantum crystals and, from a number of hot spots thus formed, the biochemical substance is detected with the use of surface enhanced Raman scattering spectra.
B82Y 20/00 - Nanooptics, e.g. quantum optics or photonic crystals
B82Y 40/00 - Manufacture or treatment of nanostructures
G01N 21/27 - ColourSpectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands using photo-electric detection
[Problem] To provide a method for producing complex crystal quantum crystals encapsulating plasmon metal quantum dots. [Solution] Quantum crystals are produced by a method characterized in that an aqueous plasmon metal complex solution formed from a ligand and a plasmon metal selected from gold, silver, copper, nickel, zinc, aluminum, and platinum is prepared and brought into contact with a carrier formed from a metal or a metal alloy having an electrode potential lower than that of the plasmon metal, or a metal that has been brought to an electrode potential lower than that of the plasmon metal, such that plasmon metal complex is precipitated and quantum crystals having a plasmon enhancement effect are disposed on the metal carrier. The metal complex crystals encapsulate metal quantum dots, and excellent subject adsorption capability, as well as an excellent surface plasmon excitation-field enhancement effect, is obtained as a result of the quantum size.
C07C 229/08 - Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated having only one amino and one carboxyl group bound to the carbon skeleton the nitrogen atom of the amino group being further bound to hydrogen atoms
An assaying substrate having a high SERS activity and a method of assay using the substrate. The substrate is a metallic substrate obtained by aggregating 5-100 nm nanoparticles (including clusters) of a metal, the nanoparticles having an SERS activity, on a metallic substrate having a less noble (higher) electrode potential than the nanoparticles and fixing the nanoparticles in an optimal aggregated state that makes the nanoparticles serve as a hot site. Since the substrate surface has a region where the metal nanoparticles having an SERS activity have been aggregated in the optimal state, adsorbing a sample to be detected onto the region and irradiating the region with given laser light bring about surface plasmon resonance on the nanoparticles in the optimal aggregated state. Because of this, surface-enhanced Raman scattering (SERS) light from the antigen sample to be detected can be detected.
patents
