WO2019245020A1 - Procédé de biopsie liquide basé sur l'autofluorescence ciblant des conjugués protéiques associés à une maladie - Google Patents

Procédé de biopsie liquide basé sur l'autofluorescence ciblant des conjugués protéiques associés à une maladie Download PDF

Info

Publication number
WO2019245020A1
WO2019245020A1 PCT/JP2019/024678 JP2019024678W WO2019245020A1 WO 2019245020 A1 WO2019245020 A1 WO 2019245020A1 JP 2019024678 W JP2019024678 W JP 2019024678W WO 2019245020 A1 WO2019245020 A1 WO 2019245020A1
Authority
WO
WIPO (PCT)
Prior art keywords
fluorescent
value
fluorescence
autofluorescence
cancer
Prior art date
Application number
PCT/JP2019/024678
Other languages
English (en)
Japanese (ja)
Inventor
裕起 長谷川
長谷川　克之
Original Assignee
有限会社マイテック
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 有限会社マイテック filed Critical 有限会社マイテック
Publication of WO2019245020A1 publication Critical patent/WO2019245020A1/fr

Links

Images

Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • 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
    • G01N21/64—Fluorescence; Phosphorescence
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • 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

Definitions

  • the present invention targets a disease-related protein conjugate, and excites its auto-fluorescence by surface plasmon enhancement to detect and adopt a fluorescent colony having a brightness equal to or higher than a predetermined threshold.
  • the present invention relates to a liquid biopsy method targeting a relevant protein conjugate.
  • a definitive diagnosis of cancer is made by a so-called biopsy (biopsy) method, in which a part of the cancer tissue is collected by puncture or endoscopic treatment, and the tissue fragment is examined for histopathology.
  • biopsy a so-called biopsy
  • Many biopsies are invasive procedures that involve risks such as bleeding and complications of bacterial infection, and biopsy may be difficult at some major sites.
  • a liquid biopsy an attempt to diagnose a disease with an accuracy comparable to that of pathological diagnosis using a sample that can be easily collected, such as blood or urine, is called a liquid biopsy (liquid biopsy). Because of its rapidity and simplicity, it has attracted much attention as suitable for clinical applications.
  • microRNA microRNA: miRNA
  • CTC circulating tumor cells
  • EMT epithelial-mesenchymal transition
  • peripheral blood circulation microRNA and exosomes containing it are expected to be particularly useful for early diagnosis.
  • microRNAs bind complementarily to messenger RNA (mRNA) before being translated into proteins in cells and inhibit the translation of the mRNA, and are important in various biological phenomena as so-called fine tuners of gene expression.
  • mRNA messenger RNA
  • fine tuners of gene expression This is because it plays a role, and it is known that in cancer cells, the microRNA expression control mechanism is disrupted and, for example, microRNAs that promote cell growth are highly expressed.
  • exosomes almost all cells secrete extracellular vesicles (exosomes), and it is said that early cancer can be diagnosed by detecting exosomes secreted by early cancer cells in body fluids. That's because.
  • the protein conjugate associated with this kind of disease is selectively captured by the plasmon metal nanocrystal by having a positive charge, and is enhanced by the surface plasmon enhancement effect of the plasmon metal nanocrystal by irradiation of excitation light, and a predetermined Raman light should be observable, but the observation was extremely difficult.
  • the cause is that in apoptotic cells, the inhibitor of CAD (caspase-activating DNase) is degraded, and the activated CAD cleaves the DNA in nucleosome units, so that the fragmented DNA is captured as a multiple of about 200 bp.
  • the present inventors have conducted intensive studies and found that, when the protein conjugate to be detected is enhanced by the surface plasmon enhancing effect of the plasmon metal nanocrystals, it emits autofluorescence having a luminance higher than a predetermined level that can be confirmed with a fluorescence microscope. (Fig. 1), and a plurality of colonies are observed as in the constellation of the night sky shown in Fig. 1. In the case of a small one, it is observed as about 25 ⁇ m, and in the case of a large one, it is observed as about 150 ⁇ m.
  • the conjugate Since the conjugate is released into blood and other body fluids by pathological cell death typified by apoptosis upon the occurrence of the disease, the conjugate was found to be deeply related to the disease. Conventionally, in this type of cell observation, autofluorescence becomes background light, and the signal-to-noise ratio (S / N ratio) of the fluorescent image is reduced. Therefore, fluorescent labeling of the target is recommended for endoscopy, and in order to obtain the best data from the fluorescence image, the signal (preferably fluorescent labeling) and the background (fluorescent labeling must be used). However, it was necessary to make the difference as large as possible (Non-Patent Document 6).
  • Non-Patent Document 7 In cell observation of a fluorescence microscope using autofluorescence, it is common to observe cells by imaging the fluorescence lifetime, which is another parameter of fluorescence, instead of the fluorescence intensity of autofluorescence.
  • Circulating Tumor Cells Circulating tumor isolation and diagnostics: toward routine clinical use.
  • Permuth-Wey J et al. A Genome-Wide Investigation of MicroRNA Expression Identifies Biologically-Meaningful MicroRNAs That Distinguish between High-Risk and Low-Risk Intraductal Papillary Mucinous, Neoplasms of the Philippines. Ellen Heitzer, Peter Ulz and Jochen B.
  • the present inventors have targeted the cell free DNA containing the remaining peripheral circulating tumor DNA (DNActDNA), and as a result of intensive studies, the protein conjugate to be detected has been found to be present in blood and other body fluids due to the occurrence of disease. Since it is released by pathological cell death typified by apoptosis, it is closely related to diseases. Moreover, in the fluorescence image, a plurality of colonies are observed as in the constellation of the night sky shown in FIG. 1. A small one shows a spread of about 25 ⁇ m, and a large one shows a spread of about 150 ⁇ m.
  • DNActDNA peripheral circulating tumor DNA
  • the problem to be solved by the present invention is to target cells free DNA containing circulating tumor DNA (ctDNA) including peripheral blood circulating tumor DNA, and to analyze cancer by histone modification analysis and chromatin structure analysis using clinical tissue samples.
  • An object of the present invention is to provide a detection method for detecting and diagnosing a tumor, which leads to the onset of the disease, quickly and easily by a liquid biopsy method using autofluorescence of a disease-related substance.
  • the present invention provides a) a sample prepared by directly or diluting a culture solution containing a body fluid or cells, with a measurement substrate having a plasmon metal nanocrystal region exhibiting a surface negative charge in a sample, thereby reducing the positive charge in the sample.
  • binarizing the brightness of the fluorescent colony and adopting a fluorescent colony having a brightness equal to or higher than a predetermined threshold is to increase the measurement accuracy by adopting only a fluorescent colony having a brightness equal to or higher than a certain threshold.
  • the fluorescence microscope can determine a threshold for binarization.
  • the fluorescent colony having the selected predetermined threshold or more is used in the calculation step.
  • the analysis range In the process of calculating the total luminance value and the area value, after acquiring the fluorescence image in the charge trapping region of the protein conjugate formed in the plasmon metal nanocrystal region on the measurement substrate, set the analysis range, and set the analysis range.
  • a fluorescent colony of a capture substance that emits fluorescence at or above a predetermined threshold based on the luminance value of the fluorescent image area is adopted, divided into at least three stages from the total luminance value or the total area value, and the cancer is determined from the area value area.
  • a risk judgment method is adopted that classifies at least three levels: risky, observation required, and low cancer risk.
  • the step of calculating the ratio of RGB of the selected fluorescent colonies having a predetermined threshold or more and / or two wavelength ratios correlated therewith in the step of calculating the charge of the protein conjugate formed in the plasmon metal nanocrystal region on the measurement substrate.
  • the analysis range After acquiring the fluorescence image in the capture area, set the analysis range, calculate the total area of the selected portions of each of the RGB based on the brightness value and circularity of the fluorescent colony in the fluorescence image area in the analysis range, A risk determination method of classifying at least three types of malignant tumors, benign tumors, and healthy persons based on the ratio values of B / G and G / R of the obtained total areas of RGB is adopted.
  • the protein conjugate to be detected as the detection target contains fragmented DNA of about 200 bp, which is the activated CAD (caspase-activated DNase) that cleaves DNA in nucleosome units.
  • a plurality of the colonies of the protein conjugate observed in the fluorescence image are observed to spread as small as about 25 ⁇ m, and as large as about 150 ⁇ m.
  • the body fluid as a specimen is plasma or serum separated from lymph or blood, and the protein conjugate contains cell free DNA (cfDNA) that binds to histone protein and exhibits a positive charge. Circulating tumor DNA (ctDNA) released from cancer cells. Further, when the specimen is an iPS cell, it is intended to identify canceration of the iPS cell.
  • cfDNA cell free DNA
  • ctDNA Circulating tumor DNA
  • a disease-related substance is selectively captured, and a fluorescent colony having a luminance equal to or higher than a predetermined threshold by adopting a surface plasmon enhancing effect of autofluorescence that usually becomes noise in fluorescence diagnosis is adopted.
  • a surface plasmon enhancing effect of autofluorescence that usually becomes noise in fluorescence diagnosis.
  • a biological protein has auto-fluorescence, and this auto-fluorescence is a signal (desired to be fluorescently labeled) background (such as auto-fluorescence not desirably labeled with fluorescence) in observing the target fluorescence. Formed and becomes noise.
  • the autofluorescence of the biological protein reflects the structure of the biological protein itself, the structural analysis using the autofluorescence is particularly effective in the following points. That is, in recent years, from the results of analyzes performed so far, during the development and progression of cancer cells, not only genomic changes but also epigenomic changes have accumulated in large numbers, and epigenome modification has been applied to chemical control and physical control. It is roughly divided.
  • H3K27ac acetylation
  • methylation of H3 lysine 27 which are characteristically observed in a region that positively activates gene expression
  • the gene expression status correlates well with the chromatin status of the promoter region where the transcription start site is located
  • the chromatin status of the enhancer is also deeply involved in the gene expression regulation specific to the cell lineage.
  • the enhancer region using H3K27ac as an index is important also in the three-dimensional structural form ⁇ (genomic topology) ⁇ of the gene. Therefore, observation of autofluorescence using clinical tissue samples suggests the possibility of early diagnosis of cancer and identification of cancer sites by histone modification analysis and chromatin structure analysis.
  • the protein conjugate to which the cell free DNA has bound binds to the histone to form a nucleosome, and when the histone protein is methylated, stabilizes the DNA in the nucleosome. Furthermore, DNA methylation abnormalities are said to cause inactivation of tumor suppressor genes and cause cancer development. Therefore, collecting and analyzing cell free DNA (cfDNA) released from cells into the blood by apoptosis, including circulating tumor DNA (ctDNA), is considered to be a very promising target for liquid biopsy. . However, it is extremely difficult to collect free DNA (cfDNA), including ctDNA in blood, and since it is in a very small amount, PCR is the basis of detection, and testing is roughly divided into mutation-specific PCR and digital PCR.
  • Non-Patent Document 5 in view of the fact that DNA usually exhibits a negative charge, strongly binds to a histone protein exhibiting a positive charge, and has the property of being captured by plasmon nanometal crystals exhibiting a negative charge, it is captured by a chip, An attempt was made to detect it by spectral spectroscopy.
  • the histone protein conjugate that binds to the DNA captured on the chip is extremely small because it is finely divided by apoptosis and is captured, so that this capture substance is appropriately excited with excitation light, and Raman is enhanced and reflected. It was extremely difficult to detect light, and it was difficult to know the result easily and quickly by the liquid biopsy method.
  • the selective capture of a disease-related substance is performed as follows. If the cell free DNA (cfDNA) to be targeted, including ctDNA, binds to histone proteins and is present in the blood as a minimum unit of nucleosomes or higher order chromatin, the DNA wrapped around histones is It exists stably by binding to histone proteins and by methylation. Moreover, as a result of showing a positive charge, a negative charge is shown on the surface in blood.
  • cfDNA cell free DNA
  • cell free DNA is released into the blood from cells of healthy subjects via apoptosis, but tumor cell free DNA is released from cancer cells, and these are methylated by themselves and histone. It is presumed that they bind tightly and form a disease-specific DNA protein conjugate from other diseased cells.
  • the DNA released from cells of these healthy subjects, cancer patients, and other diseased patients has not only genetic abnormalities but also epigenetic genetic abnormalities due to base modifications, and is released from each cell Protein conjugates can be chemically or physically different. As a result, they found that the protein conjugate formed by DNA released from cells of healthy subjects, cancer patients, and other disease patients showed a difference in the wavelength of autofluorescence.
  • DNA released into body fluids, particularly plasma or serum due to pathological degradation such as apoptosis of cells binds to histone proteins or the like to form protein conjugates.
  • FIG. 1 shows a fluorescent image obtained using an Olympus DM (dichroic mirror) 405-445 / 514, and shows a method of adopting 10 points of high luminance for each sample.
  • FIG. 2 shows 470-490 nm and 600-620 nm images obtained using Olympus DM405-445 / 514.
  • FIG. 3 is a graph showing a spectrum obtained using BS10 / 90.
  • FIG. 4 shows a silica gel drying container, in which (a) shows a state in which crystals obtained by centrifuging blood are dropped, and (b) shows a state in which plasma is dried in the container using silica gel.
  • First step A of the first method of the present invention Explanatory drawing of preparation of a measurement chip (proteo chip), Second step B: explanatory diagram of determination of analysis range of proteochip, Third step C: It is an explanatory view of quantifying the area of (the adopted fluorescent colony) of the proteochip.
  • Image acquisition step (1) ⁇ analysis range determination step (2) ⁇ fluorescent colony selection step (3) ⁇ calculation step of total area value of selected colonies in RGB fluorescent image (4) ⁇ obtained RGB of the second method of the present invention
  • the first method preferably employs the following steps.
  • Equipment used Keyence, fluorescence microscope BZ-X710
  • Light source Metal halide lamp 80W
  • Fluorescent filter BZ-X filter DAPI (460 ⁇ 25nm)
  • Analysis software BZ-X Analyzer a) Step of selectively capturing cancer-related substances: FIG. 5A A measurement substrate having a plasmon metal nanocrystal region exhibiting a surface negative charge in the sample: a proteochip (FIG. 5A (1)) is brought into contact with a specimen prepared by directly or diluting a body fluid or a culture solution containing cells (FIG. 5A).
  • the protein conjugate captured on the plasmon metal nanocrystals (about 8 mm in diameter) is irradiated with excitation light to enhance the auto-fluorescence of the captured protein conjugate by the surface plasmon enhancing effect, and the fluorescent colony is imaged (FIG. 5B).
  • a substance having a strong fluorescence (a blue substance, a binarization threshold of 13 or more based on a luminance value in the following measurement conditions) is adopted, and its area value is calculated (unit: ⁇ m 2 ). From the area value, 0 to 19999 are classified into three stages of A when the cancer risk is low, 20,000 to 29999 are required B, and 30,000 to have cancer risk.
  • FIG. 6 shows a second method of the present invention, in which a fluorescent colony having a certain threshold or more is adopted from a fluorescent image, and a ratio value is calculated based on RGB and / or a two-wavelength ratio correlated therewith.
  • FIG. 6A three fluorescent images of RGB are acquired for one sample.
  • the analysis range is determined, and the area is enclosed by the ROI (FIG. 6 (2)).
  • “circularity and luminance of fluorescent colonies” are used as thresholds, analysis conditions are determined, and dust and the like are excluded (FIG. 6 (3)).
  • the total area value of the fluorescent colonies selected from the RGB fluorescent images is calculated (FIG. 6 (4)).
  • the details are as follows.
  • the optimal excitation light and fluorescence wavelength for directly measuring the autofluorescence specific to cancer-related substances were identified. From the results of analyzing the auto-fluorescence of cancer-related substances and analyzing the fluorescence wavelength, the excitation wavelength of RGB that emits strong auto-fluorescence and the wavelength to measure the auto-fluorescence of RGB were determined.
  • An LED light source was used as a light source, and a fluorescence wavelength filter was attached to an Olympus erected microscope BX-63 to limit the fluorescence wavelength to be measured and to acquire a fluorescence image.
  • B excitation light is 375-400nm and fluorescence wavelength is 460 ⁇ 25nm
  • G excitation light is 470-495nm and fluorescence wavelength is longer than 510nm
  • the excitation light of R is 530 to 550 nm and the fluorescence wavelength is in the long wavelength range of 575 nm or more.
  • B is the excitation light and fluorescence wavelength at which autofluorescence of the cancer-related substance is strongly emitted
  • G is the excitation light and fluorescence wavelength at which autofluorescence of benign disease is strongly emitted.
  • the focus (Z axis) and the measurement position (X, Y axes) are determined.
  • the LED light source and the fluorescent wavelength filter are set to the above G
  • the focus (Z axis) is adjusted to the same measurement position (X, Y axis) as the B setting, and the fluorescent image is measured.
  • the measurement positions (X, Y axes) it is possible to acquire the respective fluorescence images of the same cancer-related substance at the same position on the chip.
  • a fluorescent image at the same measurement position (X, Y axis) is measured by using the LED light source and the fluorescent wavelength filter of the R setting.
  • Analysis method Analysis was performed using imaging software "cellSens" (manufactured by Olympus Japan Optical Co., Ltd.).
  • the analysis range is determined by surrounding the same cancer-related substance at the same position attached to the biochip of the three types of fluorescence images measured with each excitation light with the ROI, and the area value of the substance in the following RGB setting range is calculated.
  • B was set to select an adhering substance having a luminance value in the range of 28000 to 50,000 and to remove an adhering substance having a circularity of 0.3 or less.
  • G is a setting to select the deposits in the brightness range of 27,000 to 50,000 and to remove the deposits with a circularity of 0.3 or less.
  • R is a setting to select the deposits in the luminance range of 21000 to 50,000 and to remove the deposits with a circularity of 0.3 or less.
  • This luminance value setting range was set according to the fluorescence intensity of the autofluorescence of the attached substance and the light intensity of the LED light source.
  • the analysis software automatically removes irregularly shaped foreign matter and increases the analysis accuracy.
  • the range of the luminance value is in the range of 10,000 to 70,000, preferably about 20,000 to 50,000, and the range of the circularity is in the range of 0.9 to 0, preferably about 0.3. This circularity is adjusted according to the condition of the fluorescent colony.
  • the ratio values of the two wavelength ratios such as B / G and G / R were calculated from the RGB area values calculated by the measurement and analysis settings.
  • the ratio of B / G was 1.9 to 1.0 for malignant tumors.
  • the range was around 0.1 for benign tumors and 0.2 to 0.8 for healthy subjects.
  • FIG. 7 shows an image diagram of a microscope stage when four proteochips (measurement substrates) are set in a holder to automate measurement.
  • the measurement positions (X-axis, Y-axis) of the chips (1) to (4) are registered in advance, and the focus (z-axis) of the chips (1) to (4) is independently adjusted.
  • the X, Y, and Z axes are determined, three types of RGB measurement of four chips are automatically performed, and four RGB fluorescent images are obtained.
  • the body fluid which is a specimen, is not only plasma or serum separated from lymph or blood, but also contains urine, saliva, etc., and a protein conjugate is a cell free DNA (cfDNA) which binds to histone protein and shows a positive charge.
  • the cell free DNA includes circulating tumor DNA (ctDNA) released from the cells.
  • the light source for exciting the plasmon metal nanocrystal that captures the disease-related substance preferably uses excitation light having a wavelength of 405 nm, which is used to excite the tumor-affinity fluorescent substance. In this case, the fluorescence wavelength is observed around 630 nm.
  • the protein conjugate captured in the present invention is circulating tumor DNA (CtDNA), a nucleosome bound to a histone protein or a higher-ordered chromatin, and when histone is subjected to methylation modification, it can be used as a target for cancer diagnosis by the liquid biopsy method using autofluorescence.
  • CtDNA tumor DNA
  • a nucleosome bound to a histone protein or a higher-ordered chromatin a nucleosome bound to a histone protein or a higher-ordered chromatin
  • a substrate having a plasmon metal nanocrystal region used in the method of the present invention is called a proteochip.
  • the manufacturing method is as follows (see Patent Document 1). 1) Quantum crystals (nano-sized metal complex crystals) are aggregated by chemically reducing an aqueous solution of a metal complex on a metal substrate having a lower electrode potential (higher ionization tendency) than the metal forming the complex by the electrode potential difference.
  • quantum crystals of the silver complex are formed by a chemical reduction method by aggregating an aqueous solution of silver thiosulfate on copper or a copper alloy having an electrode potential (higher ionization tendency) lower than silver.
  • the concentration of the metal complex in the aqueous solution should be determined mainly in consideration of the size of the formed quantum crystal.
  • a dispersant it is better to consider the concentration, and usually 100 ppm to 5000 ppm.
  • a concentration of 500 to 2000 ppm is preferable.
  • 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) which is 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 ionic value
  • 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 excitation light.
  • the metal complex when the metal complex is a silver complex, the metal complex is preferably formed by a reaction between a silver complexing agent having a stability constant (formation constant) (log ⁇ i) of 8 or more and silver halide.
  • a silver complexing agent having a stability constant (formation 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 needle-like nanocrystals of silver oxide containing silver peroxide exhibit surface plasmon enhancing effect by irradiation with excitation light typified by laser light, and autofluorescence of cancer-related substances typified by adsorbed histones.
  • the composite needle-shaped nanocrystals of silver oxide of the present invention are those in which silver oxide containing silver peroxide is self-organized to form a neuron-shaped three-dimensional superstructure (mesocrystal) (Patent Document) 8 and 9), the silver complex aqueous solution can be formed by performing constant potential electrodeposition using an Ag / AgCl electrode or oxidizing silver quantum crystals by alkali treatment.
  • Crystals such as silver thiosulfate quantum crystals can be easily formed by alkali treatment (treatment with an aqueous solution of sodium hypochlorite). 5) Plasmonic formed by depositing a thin film of silver and zinc oxide on a substrate having a periodic structure with a pitch of 350 nm, which realizes advanced detection, sensitivity and quickness of a fluorescent label marker as long as it can adsorb disease-related substances.
  • Localized plasmon fluorescence consisting of silver nanoparticles with a uniform grain system by dispersing chips (Patent Document 2) and metal nanoparticles in an organic solvent, volatilizing the organic solvent, and self-organizing the metal nanoparticles in two dimensions. You may use the reinforcement sheet (patent document 3).
  • specimen used in the present invention A specimen is prepared from a body fluid including blood. Since erythrocytes show strong autofluorescence, it is better to centrifuge to extract only plasma. In the case of a cancer disease as a disease-related substance, the dilution ratio is determined by taking into account the charge intensities of the protein conjugate in which histone and DNA are bound to each other and the other protein conjugate by diluting 10 to 50-fold.
  • a silver oxide mesocrystal formed by oxidizing a silver thiosulfate complex quantum crystal prepared by dropping about 1000 ppm of an aqueous solution of a silver thiosulfate complex onto phosphor bronze is preferably diluted 20 to 30 times.
  • histone proteins that form a complex with methylated DNA form stable protein conjugates (Protein-bound DNA fragments: nucleosomes or chromatins) and generate a relatively strong positive charge. Show. Therefore, the plasmon metal nanocrystals exhibiting a negative charge and exhibiting a surface plasmon enhancing effect by excitation light are selectively captured, and the complex of DNA and histone protein is stable. ) It has been found that even after storage after drying and redissolving in distilled water or the like, the characteristics of the autofluorescence of the protein conjugate before drying can be reproduced.
  • iPS cells may contain cancer gene DNA, they can be used as a sample to identify and remove iPS cell cancer by the following autofluorescence.
  • Fluorescence image acquisition process the protein conjugate captured on the plasmon metal nanocrystal substrate is irradiated with excitation light to enhance the autofluorescence of the captured protein conjugate by a surface plasmon enhancing effect, and a fluorescent colony is obtained as a fluorescent image.
  • excitation light a laser light source of 405 nm excitation light, which is suitable for exciting a hematoporphyrin derivative (tumor-affinity fluorescent substance) having different accumulation / exclusion characteristics between normal tissue and diseased tissue, was used.
  • hematoporphyrin derivative tumor-affinity fluorescent substance
  • the brightness at each point is different, the brightness at each wavelength is displayed at a rate of 100% with the brightness at 470 nm, and a graph is created using the average value of 10 points as data for each sample.
  • a peak of a cancer-related substance was present at a wavelength around 610 nm.
  • the protein conjugate associated with this kind of disease is selectively captured by the plasmon metal nanocrystal due to having a positive charge, and is enhanced by the surface plasmon enhancement effect of the plasmon metal nanocrystal by irradiation with excitation light, and confirmed by a fluorescence microscope. It has been found that it emits auto-fluorescence having a luminance higher than a predetermined level (FIG. 1).
  • Non-Patent Document 5 Cell, 2016 January
  • 164 Cell-free DNA comprises an in vivo nucleosome footprint that informs its tissues-of-origin).
  • tumor malignancy leads to higher degree of necrosis, and that tumor DNA (ctDNA) is increased in the circulating blood, predictable fragmentation of plasma DNA similar to nucleosomes cleaved by nucleases.
  • Non-patent Document 4 Circulating Tumor DNA as a Liquid Biopsy for Cancer; Climinal Chemistry 2015; 61: 112-123).
  • cancer development is a genetic disease caused by abnormalities of oncogenes and tumor suppressor genes. It has been shown that cancer can also be caused by epigenetic genetic abnormalities due to base modifications. It is thought that this effect on epigenetic genes mainly affects the transcriptional control mechanism of the gene, including methylation modification of genomic DNA and acetylation modification and methylation modification of histone proteins that form a complex with genomic DNA. There is. Therefore, the detection of the conjugate of the cfDNA to the histone protein is not only capable of distinguishing a healthy person from a cancer patient by detecting not only a genetic abnormality but also an epigenetic genetic abnormality due to modification to a base. No, suggesting the discrimination of the site of cancer occurrence.
  • Cancer-related substances in serum include histones (nucleosomes) in which DNA is wound, and chromatin (fibrils) having a structure in which they are gathered into a string. Globulin is also positively charged, but its increase is up to 2 times or less compared to other cancer-related substances, whereas the substance detected by the present invention increases more than 100 times with cancer progression Therefore, an increase other than globulin indicates that a cancer-related substance has been detected, indicating that the total brightness value or area of the selected fluorescent colony is related to the stage of the cancer.
  • Example 1 A 1000 ppm aqueous solution of silver thiosulfate was prepared, and one drop of the solution was dropped on a phosphor bronze plate, allowed to stand for about 3 minutes, and the solution was blown off. As a result, SEM images revealed that quantum crystals had been formed.
  • a photograph showing various SEM images of the nanoparticle aggregate (quantum crystal) produced in Example 1 is a thin hexagonal columnar crystal having a thickness of about 100 nm, and its surface has irregularities on the order of several nm. Is expressed. No facets specific to metal nanocrystals could be confirmed.
  • the silver complex quantum crystals of the present invention are formed on a phosphor bronze plate, while silver-only nanoparticles are deposited on a copper substrate.
  • the equilibrium potential of the silver thiosulfate complex is 0.33, Since the electrode potential is equivalent to the electrode potential (0.34), only silver (0.80) precipitates on the copper substrate. In the case of phosphor bronze, the electrode potential is 0.22, which is slightly lower. It is thought that crystals of precipitated.
  • XPS measurement 25 ⁇ l of aqueous sodium hypochlorite solution was dropped on the quantum crystal substrate for 2 minutes, A recrystallized substrate was prepared, and Ag and O were subjected to XPS measurement without etching (using a model: ULVAC-PHI, Inc./PHI5000VersaProbeII (scanning X-ray photoelectron spectrometer)). For comparison, Ag of silver oxide powder and silver chloride powder was measured. On the other hand, the recrystallized substrate was etched with an argon gas cluster ion gun for 5 minutes, and Ag and O were measured by XPS. The XPS measurement results (FIGS.
  • Patent Document 1 9 and 10 of Patent Document 1 are estimated from the EDS results (FIG. 8 of Patent Document 1), and the peak around 529 eV is an O peak derived from silver peroxide (AgO), which is 530 eV.
  • the nearby peak is considered to be an O peak derived from silver oxide (Ag2O).
  • the peak around 529 eV, which is derived from silver peroxide (AgO) is larger than the peak around 530 eV, which is derived from silver oxide (Ag2O). It can be said that this indicates that silver peroxide is formed near the substrate.
  • Silver oxide has a negative charge in an aqueous solution and is reduced by light to precipitate metallic silver. Since the tendency of silver peroxide is remarkable, 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 the silver particles is obtained.
  • the nucleosome is a basic structural unit of chromatin and has a structure in which DNA is wrapped around a histone octamer composed of four types of histones (H2A, H2B, H3, H4), and histone plays a role in packaging DNA. In addition, it plays an important role in regulating the accessibility of DNA and in controlling genes. Post-translational modification of histones controls their interaction 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.
  • DNA DNA methylation is thought to be deeply involved in this chromatin structure control.
  • genomic DNA sites where DNA methylation is observed at high density the chromatin structure generally becomes strong, and transcriptional suppression and reduction of DNA mutation rate are observed.
  • genomic imprinting X chromosome inactivation
  • tumorigenesis the structural analysis of histones and chromatin is the key to prescribing the relationship with cancer, and the analysis of factors that chemically modify the histone tail has important significance.
  • the presence or absence of a cancer symptom can be determined by fluorescence image diagnosis based on the size of the crystals. Then, it is possible to determine which organ the cancer symptom is a cancer of and the progress state thereof by analyzing chemical modification of histone tail and remodeling factors. And information about how cancer develops and progresses through this chromatin remodeling event can give clinicians information about how such cancer is likely to respond to specific chemotherapeutic agents. It allows more accurate predictions and in this way chemotherapy can be reasonably designed based on knowledge of the chemosensitivity of the tumor.
  • the ratio value of the two-wavelength ratio was calculated from the fluorescent image obtained in RGB, but the fluorescent image obtained in G tends to have a high benign tumor, and the fluorescent image obtained in B Tumors tend to be high. Therefore, in the example, when the ratio value of the prostate is analyzed by G / R, a result having a range of about 2.0 for a benign tumor, 1.7 for a healthy person, and 1.76 to 1.86 for a malignant tumor is obtained. On the other hand, for the large intestine, when the Ratio value is analyzed by B / G, it has a range of 1.9 to 2.0 for malignant tumors, 0.2 to 0.8 for healthy subjects, and around 0.1 for benign tumors. It became.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Immunology (AREA)
  • Engineering & Computer Science (AREA)
  • Chemical & Material Sciences (AREA)
  • Analytical Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Pathology (AREA)
  • General Physics & Mathematics (AREA)
  • Molecular Biology (AREA)
  • Urology & Nephrology (AREA)
  • General Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • Biomedical Technology (AREA)
  • Hematology (AREA)
  • Medicinal Chemistry (AREA)
  • Food Science & Technology (AREA)
  • Biotechnology (AREA)
  • Microbiology (AREA)
  • Cell Biology (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Investigating, Analyzing Materials By Fluorescence Or Luminescence (AREA)
  • Investigating Or Analysing Biological Materials (AREA)

Abstract

La présente invention vise à fournir un procédé de détection rapide et simple qui utilise un procédé de biopsie liquide qui est basé sur l'autofluorescence d'une substance associée à une maladie. La présente invention concerne par conséquent un procédé qui comprend : a) une étape de mise en contact d'un échantillon qui est un liquide de culture qui comprend un fluide corporel ou des cellules, ou d'un échantillon qui a été préparé par dilution d'un liquide de culture qui comprend un fluide corporel ou des cellules, avec un substrat de mesure qui a une région de nanocristaux de métal plasmonique qui présente une charge de surface négative dans un échantillon et des conjugués protéiques de piégeage de charge dans l'échantillon qui présentent une charge positive sur les nanocristaux de métal plasmonique en tant que substance associée à une maladie, b) une étape d'émission de lumière d'excitation au niveau des conjugués protéiques qui ont été piégés sur les nanocristaux de métal plasmonique pour amplifier l'autofluorescence des conjugués protéiques piégés par amplification de plasmon de surface et d'acquisition d'une image de fluorescence de colonies de fluorescence, c) une étape de binarisation de la luminance des colonies de fluorescence et de sélection de colonies de fluorescence qui ont une luminance qui est supérieure ou égale à une valeur seuil prescrite, et d) une étape de calcul d'une valeur de rapport à l'aide d'une valeur de luminance totale et/ou d'une valeur de surface totale pour les colonies de fluorescence sélectionnées qui sont au niveau ou au-dessus de la valeur seuil prescrite ou à l'aide de RVB et/ou d'un rapport à deux longueurs d'onde correspondant pour les colonies de fluorescence sélectionnées qui sont au niveau ou au-dessus de la valeur de seuil prescrite.
PCT/JP2019/024678 2018-06-21 2019-06-21 Procédé de biopsie liquide basé sur l'autofluorescence ciblant des conjugués protéiques associés à une maladie WO2019245020A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
JP2018117878A JP2021156576A (ja) 2018-06-21 2018-06-21 疾病関連タンパク結合体を標的とする自家蛍光によるリキッド・バイオプシィ法
JP2018-117878 2018-06-21

Publications (1)

Publication Number Publication Date
WO2019245020A1 true WO2019245020A1 (fr) 2019-12-26

Family

ID=68983936

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/JP2019/024678 WO2019245020A1 (fr) 2018-06-21 2019-06-21 Procédé de biopsie liquide basé sur l'autofluorescence ciblant des conjugués protéiques associés à une maladie

Country Status (2)

Country Link
JP (1) JP2021156576A (fr)
WO (1) WO2019245020A1 (fr)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11609229B2 (en) 2020-04-30 2023-03-21 Mytech Co. Ltd. Fluorescence counting system for quantifying viruses or antibodies on an immobilized metal substrate by using an antigen-antibody reaction
EP4083223A4 (fr) * 2019-12-25 2024-01-17 MYTECH Co., Ltd. Procédé de biopsie liquide basé sur l'autofluorescence ciblant des nucléosomes fragmentés par apoptose

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2001340072A (ja) * 2000-03-29 2001-12-11 Matsushita Seiko Co Ltd 微生物計量装置
JP2016208959A (ja) * 2015-02-26 2016-12-15 有限会社マイテック 疾病の遠隔地画像診断システム
JP2017189626A (ja) * 2008-05-20 2017-10-19 ユニバーシティー ヘルス ネットワーク 螢光に基づく画像化およびモニタリング用装置ならびにその方法
JP2017223678A (ja) * 2013-05-08 2017-12-21 有限会社マイテック バイオチップ

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2001340072A (ja) * 2000-03-29 2001-12-11 Matsushita Seiko Co Ltd 微生物計量装置
JP2017189626A (ja) * 2008-05-20 2017-10-19 ユニバーシティー ヘルス ネットワーク 螢光に基づく画像化およびモニタリング用装置ならびにその方法
JP2017223678A (ja) * 2013-05-08 2017-12-21 有限会社マイテック バイオチップ
JP2016208959A (ja) * 2015-02-26 2016-12-15 有限会社マイテック 疾病の遠隔地画像診断システム

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
LECHARDEUR, DELPHINE: "Contrasting nuclear dynamics of the caspase-activated DNase (CAD) in dividing and apoptotic cells", THE JOURNAL OF CELL BIOLOGY, vol. 167, no. 5, 2004, pages 851 - 862, XP055665520, ISSN: 0021-9525, DOI: 10.1083/jcb.200404105 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP4083223A4 (fr) * 2019-12-25 2024-01-17 MYTECH Co., Ltd. Procédé de biopsie liquide basé sur l'autofluorescence ciblant des nucléosomes fragmentés par apoptose
US11609229B2 (en) 2020-04-30 2023-03-21 Mytech Co. Ltd. Fluorescence counting system for quantifying viruses or antibodies on an immobilized metal substrate by using an antigen-antibody reaction

Also Published As

Publication number Publication date
JP2021156576A (ja) 2021-10-07

Similar Documents

Publication Publication Date Title
US10215700B2 (en) Plasmonic chip for observing cancer related substances by localized surface plasmon resonace
KR102162706B1 (ko) 암 관련 물질의 라만 정량 방법
JP2024116149A (ja) ヌクレオソームの定量法
WO2019245020A1 (fr) Procédé de biopsie liquide basé sur l'autofluorescence ciblant des conjugués protéiques associés à une maladie
Ito et al. Silver nanoscale hexagonal column chips for detecting cell-free DNA and circulating nucleosomes in cancer patients
TWI818278B (zh) 透過自體螢光的液態生檢法
JP2016044993A (ja) ヒストン化学修飾判定による癌診断方法

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 19822648

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

32PN Ep: public notification in the ep bulletin as address of the adressee cannot be established

Free format text: NOTING OF LOSS OF RIGHTS PURSUANT TO RULE 112(1) EPC (EPO FORM 1205A DATED 09/03/2021)

NENP Non-entry into the national phase

Ref country code: JP

122 Ep: pct application non-entry in european phase

Ref document number: 19822648

Country of ref document: EP

Kind code of ref document: A1