Liquid Biopsy Method with Measuring Autofluorescence of Nucleosomes Fragmented and Released into blood from Cell Apoptosis

Abstract

[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.

Claims

1: An auto-fluorescent liquid biopsy comprising: a) a step of charge-capturing a fragmented DNA (fragmented nucleosome) as a disease-related substance in a sample comprising a body liquid or a culture liquid containing cells by using a measurement substrate having a plasmon metal meso-crystalline region; b) a step of irradiating excitation light to the captured fragmented DNA (fragmented nucleosome) on the plasmon metal meso-crystal, enhancing its autofluorescence by a surface plasmon enhancement effect, determining a fluorescence image of a fluorescence colony of the fragmented DNA (fragmented nucleosome) by determining a certain measurement region (ROI), and acquiring a fluorescence colony image of any region of B as a signal of a malignant; c) a step of adopting a pixel exhibiting a luminance equal to or higher than a predetermined threshold of the fluorescence colony image; d) a step of calculating a total area value of pixels above a predetermined threshold in the B region.

2. (canceled)

3: An auto-fluorescent Liquid Biopsy method, comprising: a) a step of charge-capturing a fragmented DNA (fragmented nucleosome) as a disease-related substance in a sample comprising a body liquid or a culture liquid containing cells by using a measurement substrate having a plasmon metal meso-crystalline region; b) a step of irradiating excitation light to the captured fragmented DNA (fragmented nucleosome) on the plasmon metal meso-crystal, enhancing its autofluorescence by a surface plasmon enhancement effect, determining a fluorescence image of a fluorescence colony of the fragmented DNA (fragmented nucleosome) by a certain measurement region (ROI), and acquiring a fluorescence colony image of any region of B as a signal of a malignant while acquiring a fluorescence colony image of any region of G as a signal of a benign; c) a step of adopting a pixel exhibiting a luminance equal to or higher than a predetermined threshold of the fluorescence colony image; d) a step of calculating a ratio value of the B region/G region or the G region/B region of the total area value of pixels collected therefrom.

4: The method of claim 3, wherein the B/G ratio range from 1.9 to 2.0 is adopted for malignancies, and the B/G ratio range from 0.2 to 0.8 is adopted for healthy individuals, the B/G ratio of about 0.1 is adopted for benign.

5: The method of claim 1, wherein the captured fragmented DNA (fragmented nucleosome) comprises a methylated cancer suppressor gene, a hypermethylated RB gene, and p16 in addition to p14, p53, etc.

6: The method of claim 1, further comprising: e) a step of measuring a spectroscopic spectrum as an additional data other than the total area value.

7: The method of claim 3, further comprising: e) a step of measuring a spectroscopic spectrum as an additional data other than the ratio value.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0039] FIG. 1 shows a fluorescence image obtained using an Olympus DM (Dichroic Mirror) 405-445/514, where 10 points of high brightness for each sample are adopted.

[0040] FIG. 2 shows 470-490 nm and 600-620 nm images acquired using an Olympus DM405-445/514.

[0041] FIG. 3 is a graph showing spectral spectra obtained using a BS10/90.

[0042] FIG. 4 shows a silica gel drying vessel, (a) shows a dropping state of a crystal obtained by centrifuging blood, and (b) shows a state in which plasma is dried in a container with silica gel.

[0043] FIG. 5A shows the first step A of the first method is an explanatory view of the preparation of a measuring chip (proteo chip).

[0044] FIG. 5B shows the second Step 2 B of Illustration of the determination of the analytical scope of the proteochip.

[0045] FIG. 5C show the third Step 3 C of Illustration of the numerical representation of the area of the (adopted fluorescent colony) of the proteochip.

[0046] FIG. 6 shows the image acquiring step of the second method of the present invention, where (1) analysis range determining step, (2)fluorescent colony adopting step, (3) calculation step of the total area value of the selected colonies in OOARGB fluorescent image, (4) is an explanatory diagram showing a step of obtaining RGB total area and (5) preparing a Ratio value such as B/G,G/R from.fwdarw.obtained RGB total area.

[0047] FIG. 7 is an explanatory view of a microscope stage employed when the method of the present invention is automatically performed.

[0048] FIG. 8 shows a conceptual diagram of the inventive Ratio calculation process.

[0049] FIG. 9 is an explanatory view of the analysis method when the fragmented nucleosome which exists in the centrifuged supernatant liquid (plasma portion) is measured the autofluorescence using the proteo chip of the present invention.

[0050] FIG. 10 is a graph showing distributions of Ratio values of healthy subjects, benign tumors, and malignancies that can be identified by the method of FIG. 9, wherein the y-axis height represents the ratio of B region/G region, and the x-axis represents the sample count.

[0051] FIG. 11 is a comparative illustration of multi-stage carcinogenesis hypothesis models comparing 1) the inventive test levels for detecting fragmented nucleosomes with 2) the test levels by using the current PET-CT, CT, and MRI.

DETAILED DESCRIPTION OF THE INVENTION

[0052] In the implementation of the liquid biopsy method according to the present invention, the first method is as follows

[0053] In the first method, the fragmented DNA (fragmented nucleosome) captured on the substrate is excited by 1) a single wavelength excitation light from a laser or 2) an excitation light of a fixed wavelength width adopted by a filter, and the total area of pixels above a predetermined threshold of the fluorescent colony is measured.

[0054] Equipment used: Fluorescence microscopy

[0055] BZ-X710 made in KEYENCE CORPORATION,

[0056] light source: Metal halide lamp 80 W

[0057] Fluorescent Filter: BZ-X Filter DAPI (460±25 nm)

[0058] Analysis Software: BZ-X Analyzer

[0059] a) Selective capture steps for cancer-related agents:

[0060] as shown in FIG. 5A

[0061] Measurement substrate having plasmon metal meso-crystalline regions exhibiting surface negative charges in a sample: A proteo chip (FIG. 5A (1)) is contacted with a specimen prepared by diluting a body fluid or a culture solution containing cells as it is or by a diluted culture solution (FIG. 5A (2)), and a protein conjugate exhibiting positive charges in the specimen is trapped by the plasmon metal meso-crystal as a disease-related substance (FIG. 5A (3)).

[0062] b) Fluorescence image acquisition process:

[0063] (1) Placing the Proteo chip with cancer-related substances on the fluorescence microscope.

[0064] (2) Determining the measurement position (X and Y axes) of the chip while viewing the bright-field image, and click the auto focus button to adjust the focus (Z axis, focus) of the chip.

[0065] (3) The measurement setting is switched to BZ-X filtering DAPI, and the measurement of the fluorescent images is started. Excitation light is applied to the protein conjugate captured on this plasmon metal meso-crystal (about 8 mm in diameter), and autofluorescence of the captured protein conjugate is enhanced by Surface Plasmon Effect.

[0066] (4) Step of enhancing by the enhancement effect and acquiring the fluorescent colonies as fluorescent images (FIG. 5B);

[0067] c) adoption step of the fluorescent colonies:

[0068] Step of binarizing the luminances of fluorescent colonies within the analysis range (5 mm in diameter) and selecting fluorescent colonies with luminances equal to or higher than a predetermined threshold

[0069] d) Calculation process:

[0070] Calculating the total area (FIG. 5C) of fluorescent colonies above the selected thresholds. Here, substances with strong fluorescence (blue deposits, based on luminance values under the following measurement conditions)

[0071] Then, a binarization threshold value of 13 or more is adopted, and the area value thereof is calculated (unit μm2).

[0072] It is judged by classifying into three stages: when 0 to 19999 from the area value is A (the gun risk is low), 20000 to 29999 is observation required B and more than

[0073] 30000 is with gun risk.

[0074] FIG. 6 is a second method of calculating a ratio at a two-wavelength from the RGB and/or the related value of RGB from that adopts a fluorescent colony above a certain threshold from a fluorescent image. In FIG. 6 (1), three fluorescence images of RGB are acquired for one sample. Next, an analysis range is determined and surrounded by an ROI (FIG. 6 (2)). Here, “the circularity and luminance of the fluorescent colony” is used as a threshold value, and analysis conditions are determined to exclude dust and the like (FIG. 6 (3)). Thereafter, the total area value of the fluorescent colonies adopted from the fluorescence images of RGB is calculated (FIG. 6(4)) The details are as follows.

[0075] Typical fluorescence measurements are made using fluorescent dyes, which are observed at the fluorescence wavelengths unique to the dye. The excitation and fluorescence wavelengths are determined by the type of dye. Fluorescence measurements using dyes look at the fluorescence of the dye and are not a measure of the autofluorescence of the substance itself.

[0076] On the other hand, the autofluorescence in this invention is thought to be emitted by chemical composition and chemical structure of the substance itself, functional groups, modified compounds, biological structures, etc. In other words, each autofluorescence has an intrinsic fluorescence wavelength. The present invention looks at the intrinsic autofluorescence of cancer-related substances. It is a method to measure wavelengths of autofluorescence inherent to cancer-related substances rather than wavelengths of common dyes from multiple excitation light. We identified the optimal excitation and fluorescence wavelengths for direct measurement of cancer-related substance-specific autofluorescence. From the results of spectroscopic analysis of autofluorescence of cancer-related substances, the wavelengths for measuring the excitation light of emitted RGB and the autofluorescence of RGB were selected and determined. An LED light source was used as the light source, and fluorescence wavelength filters were attached to the Olympus erect microscopy BX-63 to limit the fluorescence wavelength to be measured, and fluorescence images were acquired.

[0077] Specifically, the excitation light of B is in the range of 375 to 400 nm, the fluorescence wavelength is in the range of 460±25 nm, the excitation light of G is in the range of 470 to 495 nm, and the fluorescence wavelength is in the long wavelength range of 510 nm or more. The excitation light of R is in the range of 530 to 550 nm, and the fluorescence wavelength is in the range of long wavelength of 575 nm or more. The B is the excitation light and fluorescence wavelength that the autofluorescence of cancer-related substances while the G is the autofluorescence of benign diseases.

[0078] According to the second method, the present invention comprises (1) a step of setting the biochip with the cancer-related substances on the Olympus erect microscopy BX-63, and (2) a step of setting the LED light source and the fluorescent wavelength filter to the setting of B above. (3) focusing (Z-axis) while viewing the real-time fluorescence image of the biochip. When the focus (Z-axis) and measurement position (X and Y-axis) are determined, firstly the fluorescence image of the setting of B is measured. Next, the LED light source and the fluorescence wavelength filter is set to the setting of G, and the focus (Z-axis) and the same measurement position as the previous B setting (X, Y-axis) are adjusted, thereby we can obtain the data of fluorescence images of each of the same cancer-related materials at the same location on the chip can be acquired. The same process is applied to measure the fluorescence image at the same measurement position (X and Y axes) with the LED light source and fluorescence wavelength filter of R setting in the same way.

Analytical Methods

[0079] Analysis was performed using imaging software “cellSens” (Nippon Olympus Optics). [0080] (1) Determine the analysis range by surrounding the same cancer-related substance at the same position attached to the biochip of the three kinds of fluorescence images measured by each excitation light with ROI, and calculate the area value of the substance in the set range of RGB below, wherein B is a range to be selected from the range of luminance values 28000 to 50000 and selected to remove deposits having a degree of circularity of 0.3 or less. G is a range selected from the luminance value from the range of 27000 to 50000, and also selected to remove deposits with a circularity of 0.3 or less. R is a range from the range of luminance values 21000 to 50000 and also selected to remove deposits with a circularity of 0.3 or less.

[0081] The luminance or brightness value setting range is selected depending on the fluorescence intensity of the autofluorescence of the adhering substance or the intensity of the light of the LED light source. In addition, the analysis software automatically removes foreign matter having a strange shape by analyzing with a circularity of 0.3, thereby improving the accuracy of the analysis. In the range of 10000 to 70000 and preferably in the range of 20000 to 50000 with a degree of circularity

[0082] The range is from 0.9 to 0.1, preferably on the order of about 0.3. This circularity is adjusted according to the context of the fluorescent colony. Ratio values of two-wavelength ratios, such as B/G and G/R, were calculated from the area values of RGB calculated by these measurements and analysis settings. In the case of colon, malignant tumors is ranged from 1.9 to 1.0 in Ratio values of B/G, and benign tumors is around 0.1 for and healthy individuals is ranged from 0.2 to 0.8.

[0083] It ranged from.

[0084] By performing fluorescence measurement by dividing the excitation light in this manner, it is possible to obtain a fluorescence image of stronger autofluorescence in each excitation light. By measuring and analyzing the same cancer-related substance at the same position on the biochip with different excitation light and fluorescence wavelengths, more substance-specific autofluorescence can be extracted.

[0085] The accuracy of the data could be improved.

[0086] FIG. 7 is an image diagram of a microscope stage when four proteo chips (measurement substrates) are set in a holder to automate the measurement. The measurement positions (X-axis and Y-axis) of Tip (1)˜(4) are decided in advance, and the focuses (z-axis) of the chips (1) to (4) is adjusted by hand. Once the X, Y and Z axes are determined, and RGB of four chips are automatically measured to obtain four RGB fluorescent images.

[0087] The body fluid being the analyte is not only plasma or serum separated from the lymph or blood, but also includes urine, saliva, and the like, and the protein conjugate includes cell free DNA (cf DNA) which binds to histone proteins and exhibits positive charges, and the cell free DNA includes circulating tumor DNA (ct DNA) released from the cells. Preferably, the light source that excites the plasmonic metal meso-crystal that captures the disease-related material is used as an excitation light at a wavelength of 405 nm that is used to excite the tumor-affinity fluorophore. In this case, since the fluorescence wavelength can be observed around 630 nm in that case, it is preferable to focus on the peak wavelength around 630 nm as a characteristic of the autofluorescence of the protein conjugate of the cancer-related substance even in the present invention. The protein conjugate captured here is a nucleosome containing circulating tumor DNA (ct DNA) bound to a histone protein or a chromatin with a higher order, and when the histone undergoes methylation modification, the cancer diagnosis of the liquid biopsy method by autofluorescence is performed as a target.

[0088] Chip with plasmonic metal meso-crystals exhibiting surface negative charges in the sample

[0089] A substrate having a plasmonic metal meso-crystalline region used in the method of the present invention is referred to as a proteo chip made by Mytech. Co. Ltd. The manufacturing method thereof is as follows (see Patent Document 1) [0090] 1) An aqueous solution of a metal complex is chemically reduced on a metal substrate having an electrode potential (having a larger ionization tendency) higher than that of the metal forming the complex by an electrode potential difference to aggregate quantum crystals (nano-sized metal complex crystals). In the case of a silver complex, a quantum crystal of the silver complex is formed by aggregating an aqueous silver thiosulfate solution on copper or a copper alloy having an electrode potential (having a large ionization tendency) higher than Ag by a chemical reduction method. Specifically, the concentration of the metal complex in aqueous solution should be determined primarily by considering the size of the quantum crystals to be formed, and when a dispersant is used, its concentration may also be considered, and usually can be used in the range of 100 ppm to 5000 ppm, but a concentration of 500 to 2000 ppm is preferred to prepare a nano-size crystal to be nanoclusters depending on the function of the ligand. [0091] 2) The metal complex forming the quantum crystal is selected so as to have a complex stability constant (log β) or more represented by formula (I) which correlates with the electrode potential E of the supported metal

E°=(RT/|Z|F)ln(βi)  Equation (I):

[0092] wherein E° represents the standard electrode potential, R represents the gas constant, T represents the absolute temperature, Z represents the ionic number, and F represents the Faraday constant.

[0093] Here, when the metal complex is a complex of a plasmonic metal selected from Au, Ag, Pt or Pd, it has a localized surface plasmon resonance enhancing effect by the excitation light. Stability constant (formation constant) particularly when the metal complex is a silver complex, it is preferable that (log β i) is formed by the reaction of a silver complexing agent having 8 or more and silver halide, and the silver chloride is preferred as the silver halide, and the complexing agents is preferred to be selected from the group consisting of thiosulfate, thiocyanate, sulfite, thiourea, potassium iodide, thio-salicylate, and thio-cyanurate are preferred as the complexing agent. Silver complex is flat and comprises a quantum dot consisting of nanoclusters with a uniform diameter of 5-20 nm, and the size of the quantum crystal becomes 100-200 nm. [0094] 3) The plasmon metal meso-crystal in the proteo chip used in the present invention is an oxide of the quantum crystal of a plasmon metal complex, which is a negative charge necessary for capturing a methylated nucleosome positively charged in blood by an electric charge, and which exhibits a surface plasmon enhancing effect by irradiation of the excitation light and has an effect of enhancing autofluorescence of the captured methylated nucleosome. In the case of silver complex quantum crystals, alkali treatment (for example, treatment with sodium hypochlorite aqueous solution) seems to form needle-like nanocrystals of silver oxide complex containing silver peroxide as nuclei by the following reaction (as shown in FIG. 5 of Patent Document 1). In addition, the (−) charge is charged in water, while the histone around which the DNA is wound is charged (+).

[0095] As shown in (FIG. 7(a) of Patent Document 1), it was found to selectively adsorb positively charged cancer-related substances represented by this free nucleosome. Moreover, the needle-like or acicular nanocrystals of silver oxide containing silver peroxide exhibit surface plasmon enhancement effect by irradiation with the excitation light represented by a laser light and a filtered LED light, and have been found to be preferable for detecting autofluorescence of cancer-related substances represented by adsorbed histones.

Na.sub.2S.sub.2O.sub.3+4NaClO+H.sub.2O.fwdarw.Na.sub.2SO.sub.4+H.sub.2SO.sub.4(2NaHSO4)+4

NaCl

Ag.sup.++NaCl.fwdarw.AgCl+Na.sup.+

Ag.sup.++3NaOCl.fwdarw.2AgCl+NaClO3+2Na.sup.+

Ag.sup.++OH.sup.−.fwdarw.AgOH

2Ag.sup.++2OH.sup.−.fwdarw.Ag2O+H2O [0096] 4) The complex acicular nanocrystals of silver oxides of the present invention form neuronal three-dimensional superstructures (meso-crystals) by self-assembly of silver oxides containing silver peroxide.

[0097] Therefore, as shown in (FIGS. 8 and 9 in Patent Document 1), it is possible to form the meso-crystals from a Ag ion aqueous solution by performing a potentiostatic deposition using a Ag/AgCl electrode, or by oxidizing a quantum crystal of silver by alkali treatment, the meso-crystal had better be formed from the silver complex quantum crystals, for example, silver thiosulfate quantum crystals by alkali treatment (treatment with sodium hypochlorite aqueous solution). [0098] 5) As long as the disease-related substance can be adsorbed, it is possible to use the plasmonic chip disclosed (Patent Document 2) in which a thin film of silver and zinc oxide is formed on a substrate having a periodic structure with a pitch of 350 nm which realizes high detection, sensitivity, and rapidity of a fluorescent labeling marker, or a localized plasmonic fluorescence enhancement sheet disclosed in (Patent Document 3) formed of silver nanoparticles having a uniform particle system by dispersing the thin film of silver and zinc oxide in an organic solvent and self-assembling the metal nanoparticles in a two-dimensional direction by volatilizing the organic solvent.

[0099] “Sample for Use in the Invention”

[0100] A sample is prepared from a body fluid which contains blood. Because red blood cells exhibit strong autofluorescence, it is better to centrifuge to get only plasma from blood. In the case of cancer disease as a disease-related substance, a dilution rate is determined to facilitate measurement of autofluorescence of fragmented DNA (subdivided nucleosomes) by 10 to 50-fold dilution. Among them, 20 to 30-fold dilution is preferable for silver oxide meso-crystals formed by alkali treatment of silver thiosulfate complex quantum crystals prepared by dropwise addition of about 1000 ppm of aqueous silver thiosulfate complex on phosphor bronze. In the case of cells, it is better than a mechanical disruption because it does not alter their physical properties. Advantageously, fragmented DNAs (fragmented nucleosomes) are stable protein conjugates (Protein-bound DNA fragments: nucleosome or chromatin) to form and exhibit relatively strong positive charges. Therefore, since it is selectively captured in plasmonic metal meso-crystals which exhibit a negative charge and exhibit a surface plasmon enhancement effect by the excitation light, and moreover, the fragmented DNA (subdivided nucleosomes) is stable, it is possible to reproduce the characteristics of the autofluorescence of the protein binding body before drying, even when stored after drying in a vacuum or desiccant (silica gel) and redissolved in distilled water or the like. In desiccant (silica gel) drying, this realizes the detection, not only from on-site blood collection and collection of plasma by centrifugation, but also for drying in a container, as shown in FIG. 4, so that an examination request by mailing can be made. In addition, all diseases are often caused by the accumulation of abnormal proteins; for example, the common features of neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, and Huntington's disease are due to the accumulation of aggregated abnormal proteins in neuronal cells. Abnormal proteins are cytotoxic and therefore cause neuronal degeneration and cell death. Although most aberrant proteins are marked by ubiquitination and degraded in the proteasome, the proteasome is unable to destroy aggregated aberrant proteins, and the accumulation of aggregates of ubiquitinated aberrant proteins in neuronal cells in neurodegenerative diseases suggests that the detection of aberrant proteins is possible using the Liquid Biopsy method for the detection of disease-related substances other than cancer. In addition, since the iPS cells may contain cancerous gene DNAs, the iPS cells can also be used as a sample to discriminate and remove cancelation of iPS cells by the inventive autofluorescence method.

[0101] “Fluorescence Image Acquisition Process”

[0102] Here, the fragmented DNA (fragmented nucleosome) captured on the plasmonic metal meso-crystalline substrate is irradiated, and the autofluorescence of the captured fragmented DNA (fragmented nucleosome) is enhanced by the surface plasmon enhancement effect, and the fluorescent colonies are acquired as a fluorescent image.

[0103] As then excitation light, a laser light source of 405 nm excitation light, which is said to be suitable for exciting hematoporphyrin derivatives (tumor affinity fluorescent substances) having different integration and excretion characteristics in normal and diseased tissues, was used. Protein conjugates were collected and centrifuged, and the resulting plasma was diluted 30-fold with distilled water for use.

[0104] c) “Fluorescent colony selection process”

[0105] With Olympus DM (Dichroic Mirror) 405-445/514

[0106] Since the result of FIG. 1 was obtained, for example, 10 points of high luminance for each sample (the number of points to be collected is determined according to the disease) were extracted, a circular ROI was created in the vicinity of the center, and spectral data was calculated. The 10-point adoption of fluorescent colonies is binarized with dedicated soft “cellSens”, and fluorescent colonies with brightness above predetermined thresholds are adopted.

[0107] Other image acquisition conditions are as follows.

[0108] Laser: 405 nm 50%

[0109] Objective lens: 10 times (MPLFLN10×)

[0110] Pinhole diameter: 500 nm

[0111] Wave length: 460-504 nm

[0112] slit width: 4 nm

[0113] Step: 2 nm

[0114] Resolution: 1024×1024

[0115] Averaging: 4 times

[0116] d) “Operation step”

[0117] The total area value of the pixels of the fluorescent colonies above the selected predetermined thresholds, or the ratio value by the total area ratio of the pixels in the two-wavelength region corresponding to RGB is measured.

[0118] As a result, Healthy subjects, Benign and malignant tumors can be distinguished from the total area value and the ratio value of the total area in different wavelength regions.

[0119] The ratio of the two wavelengths of R, G, and B and the ratio of the two wavelengths correlated therewith are used for identification of the healthy subjects, benign tumors and malignancies.

[0120] Images of 470-490 nm and 600-620 nm were acquired using an Olympus DM405-445/514, and the results of FIG. 2 were obtained as a result of checking whether there was a change in Ratio values. For diagnosis of prostate, G/R for benign tumor is around 2.0, healthy subjects is around 1.70, while malignancy is over 1.76 up to 1.86, which indicates a relationship with stage. As a result, the data accuracy can be improved by increasing the number of samples. In addition, since the luminance differs for each point, the luminance at each wavelength was displayed at a ratio of 470 nm as 100% luminance, and a graph was prepared with the average value of 10 points as data for each sample. Further, it was confirmed that a peak of a cancer-related substance was present at a wavelength of about 610 nm as shown in FIG. 3.

[0121] “Self-Fluorescence Images of a Target Object and a Capture Target Object Observed by its Fluorescence Microscope”

[0122] The present invention is intended for detection of fragmented DNA (fragmented nucleosomes) that occurs abnormally in cancerous and other conditions. Protein conjugates associated with this type of disease preferentially trap in plasmonic metal meso-crystals because of their positive charge

[0123] It has been found that the emission of the excitation light is enhanced by the surface plasmon enhancement effect of the plasmon metal meso-crystal and emits autofluorescence having a luminance equal to or higher than a predetermined luminance which can be confirmed by a fluorescence microscope (FIG. 1). These protein conjugates are released by pathological cell death typified by apoptosis into blood and other body fluids by the occurrence of disease, and a plurality of colonies are observed like the constellation in the night sky shown in FIG. 1, and are observed as a spread of about 25 μm in small ones and a spread of about 150 μm in large ones. Because inhibitors of CAD (caspase-activating DNase) are degraded in apoptotic cells and activated CAD cleaves DNA in nucleosome units, they are captured as fragmented DNA (fragmented nucleosomes) that are roughly 200-bp-fold higher. In detail, cell-free DNA (cf DNA) in human plasma is released into the blood as a protein binding associated with histones or TFs, preferentially survives, and is derived primarily from myeloid lymphoid cell lineages in healthy individuals, but a contribution from one or more additional tissues has been considered in certain pathologies, revealing a nucleosome footprint (footprint) that infers cell types from cfDNA in pathological conditions such as cancer and informs the origin of that tissue (non-patent literature 5: Cell, 2016 Jan. 14; 164: Cell-free DNA comprises an in vivo nucleosome footprint that informs its tissues-of-origin). It has been reported that the tumor grade leads to a higher degree of necrosis and that tumor DNA (ct DNA) is increased in the circulation, that plasma DNA exhibits a predictable fragmentation pattern similar to nuclease-cleaved nucleosomes, that cf DNA size-distribution can be assessed in healthy individuals and cancer patients, and that cf DNA in plasma is also reported to be involved in tumorigenesis and metastatic progression, revealing the importance of cf DNA as a diagnostic biomarker for liquid biopsy (non-patent literature 4: Circulating Tumor DNA as a Liquid Biopsy for Cancer; Clinical Chemistry 2015; 61: 112-123).

[0124] Now, it is a widely accepted fact that the development of cancer is a genetic disease caused by abnormalities in oncogenes and tumor suppressor genes, and the sequence of nucleotides, it has become clear that not only genetic aberrations due to mutations and deletions, but also epigenetic genetic aberrations due to modifications to bases can lead to the development of cancer. The effects on this epigenetic gene are thought to primarily affect the transcriptional control mechanisms of the gene, including methylation modification of genomic DNA and acetylation and methylation modification of histone proteins in complex with genomic DNA.

[0125] Thus, the binding of this cf DNA to histone proteins is detected not only by genetic aberrations but also by detecting epigenetic genetic aberrations due to modifications not only to distinguish healthy individuals and cancer patients, but also to suggest the distinctiveness of the cancer site.

[0126] (Selective Capture of Cancer-Related Substances)

[0127] As a cancer-related substance in serum, the fragmented DNA (fragmented nucleosomes) contains histones (nucleosomes) in which DNA is wrapped, and chromatin (fibers) in a structure in which they gather and form a string. Globulins are also positively charged, but the increase is up to 2-fold or less compared to other cancer-related substances, whereas the substance detected in the present invention increases more than 100-fold or more as cancer progresses, suggesting that an increase other than globulins indicates that cancer-related substances are being detected, and that a total area of pixels above a certain threshold of the selected fluorescent colony is associated with the stage of cancer.

[0128] Embodiments of the present invention will be described in detail below with reference to the drawings.

Example 1

[0129] A 1000 ppm aqueous solution of silver thiosulfate was prepared, and one drop of it was added dropwise onto a phosphor bronze plate.

[0130] When the solution was left to stand for about 3 minutes and blown off, a quantum crystal was produced which was confirmed by an SEM image. In a photograph (refer to FIG. 1 of Patent Document 1) showing various SEM images of the nanoparticle aggregates (quantum crystals) produced in Example 1, the crystal is a thin hexagonal prism of about 100 nm, and has unevenness on the order of several nm appears on the surface. Facets peculiar to metal nanocrystals could not be confirmed. In a photograph (FIG. 6 in Patent Document 1) showing the relationship between the standing time and the quantum crystal shape after dropping on a phosphor bronze hill, first, a hexagonal quantum crystal. In the graph (FIG. 4 of Patent Document 1) showing the result of EDS spectrum (elemental analysis) of the quantum crystal, in which the crystal formed on the phosphor bronze plate had an element derived from silver and a complex ligand, when a 1000 ppm aqueous solution of silver thiosulfate was prepared on the copper plate, one drop of the solution was dropped, left to stand for about 3 minutes, and the solution was blown off, only silver was detected and the complex ligand was not detected.

[0131] (Work of Quantum Crystals)

[0132] In the case of a 1000 ppm silver thiosulfate complex aqueous solution, when dropped on a phosphor bronze plate and left for 3 minutes, it was confirmed from the SEM image that the quantum crystal was formed in a hexagonal prism shape of around 100 nm, and that each hexagonal prism-shaped quantum crystal had roughness on the order of several nm, but facets peculiar to the metal nanocrystal could not be confirmed, and the elements derived from silver and complex ligands were detected by EDS elemental analysis, so it is presumed that the whole is a nanocrystal of a silver complex, and the roughness appearing on the surface is spread by forming quantum dots as clusters of silver in the complex. Looking at the phenomenon in which the silver complex quantum crystal is formed on the phosphor bronze plate of the present invention, while the nanoparticles of only silver are precipitated on the copper substrate, since only silver (electrode potential 0.80) is precipitated on the copper substrate because the equilibrium potential of the silver thiosulfate complex is 0.33, which is equivalent to the electrode potential of copper (0.34), and in the case of phosphor bronze, the electrode potential is 0.22, which is slightly lower, it is considered that the crystals of the silver complex are precipitated. Therefore, it seems important that 1) the complex aqueous solution is in the dilute region of 500-2000 ppm, 2) the electrode potential of the supported metal is slightly base to the equilibrium potential of the metal complex aqueous solution, and 3) the metal complex is aggregated at the electrode potential difference in order to prepare the quantum crystal. In addition, the same result was obtained when an aqueous solution of 1000 ppm thiourea silver complex was used.

[0133] (Consideration of Meso-Crystals of Silver Oxide: No. 1)

[0134] When a 5% sodium hypochlorite aqueous solution is dropped onto the quantum crystal substrate and the substrate is removed by treatment for 2 minutes, the crystal structure shown in FIG. 11 of Patent Document 1 is observed, and needle-shaped crystals and la are observed.

[0135] Since rugby ball-shaped lumps and a large lump were observed, when the respective compositions are analyzed by EDS spectrum (elemental analysis), it is considered that the needle-shaped crystals from the following reaction equation are both composed of a composite crystal of silver chloride and silver oxide, because the result of FIG. 7 in Patent Document 1 shows no chlorine and therefore silver Ag and oxygen 0 are dominant.

Na.sub.2S.sub.2O.sub.3+4NaClO+H2O.fwdarw.Na.sub.2SO.sub.4+H.sub.2SO.sub.4+4NaCl  (1)

Ag.sup.++NaCl.fwdarw.AgCl+Na.sup.+  (2)

Ag.sup.++3NaOCl.fwdarw.2AgCl+NaClO.sub.3+2Na.sup.+  (3)

Ag.sup.++OH.sup.−.fwdarw.AgOH  (4)

2Ag.sup.++2OH.sup.−.fwdarw.Ag.sub.2O+H.sub.2O  (5)

[0136] Therefore, in the formation of meso-crystals according to the present invention, silver ions and thiosulfate ions are presumed to be caused by an alkali oxidation reaction in the presence of chloride ions, but only silver oxide is formed in an ordinary aqueous solution, but it is presumed that silver peroxide is dominantly formed from the following XPS measurement.

[0137] (Consideration of Silver Oxide Meso-Crystals: No. 2) XPS Measurements:

[0138] To the quantum crystal substrate, 25 μl of a sodium hypochlorite aqueous solution was dropped for 2 minutes to form the meso-crystal substrate, and Ag and O were XPS-measured without etching the recrystallized substrate (model: ULVAC-FI Co., Ltd./PHI5000VersaProbeII (scanning X-ray photoelectron spectroscopy analyzer)). Further, for comparison of object, the Ag of the powder of silver oxide and the powder of silver chloride were 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 results of the XPS measurements (FIGS. 9 and 10 of Patent Document 1) and from the results of the EDS (FIG. 8 of Patent Document 1), it is recognized that the peak at around 529 eV is an O peak derived from silver peroxide (AgO), and the peak at around 530 eV is an O peak derived from silver oxide (Ag2O). When etched, the oxygen content decreases, but it can be said that the O peak derived from silver peroxide (AgO) at a peak around 529 eV is larger than the O peak derived from silver oxide (Ag.sub.2O) at a peak around 530 eV, which indicates that silver peroxide is formed in the vicinity of the substrates. It is presumed that the catalytic action at the time of meso-crystal formation and the electrode potential of the substrate are influenced.

[0139] Incidentally, EDS-measurement using the recrystallized substrate model: was performed by using JEOL Corporation/JSM-7001F (field emission analytical scanning electron microscopy).

[0140] Further, even when the quantum crystals of silver thiosulfate were treated with an aqueous solution of hypochlorous acid, an aqueous solution of 0.01 N caustic soda, an aqueous solution of 0.01 N hydrochloric acid, and an aqueous solution of 0.1 mol sodium carbonate, the similar results could not be obtained. Therefore, the formation of the needle-like crystals may be caused by the above reaction in the presence of silver ions and thiosulfate ions. Silver oxide is negatively charged in aqueous solution and may be reduced by light to precipitate metallic silver.

[0141] Since the tendency of silver peroxide is remarkable, it seems that it adsorbs positively charged cancer-related substances, and moreover, the surface plasmon enhancement effect between adsorbed cancer-related substances and silver particles can be obtained.

[0142] “Imaging of Fragmented DNA (Fragmented Nucleosomes)”

[0143] Nucleosomes are the basic blocks of chromatin, consisting of four histones (H2 A, H2B, H3, H4) wrapped around a histone octamer, but histones play an important part in regulating the accessibility of DNA as well as in the control of DNA. Post-translational modifications of histones control their interactions with DNA and other nuclear proteins, thus affecting reversible gene expression. Methylation, acetylation, phosphorylation, ubiquitination, SUMO, citrullination, and ADP-liposylation are the major types of histone modifications known. Depending on which sites in the histone sequence are subject to these modifications, surrounding gene expression can be activated or repressed. When various histone code-related antibodies (Genetex anti-histone antibodies) are used to examine the histone code hypothesis by observing the modified state of the histone code captured by the plasmonic chip on fluorescent images, it is possible to examine the histone code hypothesis.

[0144] It is believed that DNA methylation is deeply involved in this chromatin structure control. Genomic DNA sites with high levels of DNA methylation generally show robust chromatin organization, with transcriptional repression and reduced DNA mutation rates. There are also clear correlations between genomic DNA methylation patterns and genomic imprinting, X-chromosome inactivation, and cellular tumorigenesis. Therefore, the structural analysis of histones and chromatin in the fragmented DNA (fragmented nucleosomes) is a key to the association with cancer. So, analysis of factors that chemically modify histone tails has important implications. Using the plasmonic chip of the present invention, by trapping the fragmented DNA (fragmented nucleosome) as a cancer-related substance, the presence or absence of the cancer symptom can be judged by the fluorescence imaging diagnosis by the amount of the fragmented DNA captured on the crystal. Then, it is possible to determine the extent to which the cancer symptom is, the cancer comes from which organ and the progression state is by analyzing chemical modification and remodeling factor of the histone tail. Therefore, if a laser beam with a wavelength of 445 nm is used as the spectroscopic condition, and the “BS10/90” manufactured by Miller Olympus for Laser is used, and the spectroscopic spectrum is measured in 5 nm increments in the range of 455 to 655 nm, a peak is observed in the vicinity of 515 nm in the sample collected from the blood of a colorectal cancer patient, and the peak cannot be observed in the sample collected from the blood of a healthy person, and it is considered that it is possible to determine which organ cancer is detected by spectroscopy alone, or by considering the total area of pixels with a predetermined threshold or more in the RGB region, or the ratio G/G,G/B of the total areas of pixels with a predetermined threshold or more in the two wavelength regions in the RGB region. Then, information on how cancer develops and evolves through this chromatin remodeling event allows clinicians to more accurately predict how such cancers are likely to respond to specific chemotherapeutic agents, and in this way, chemotherapy based on knowledge of tumor chemosensitivity can be rationally designed.

[0145] In the present embodiment, Ratio value of the total area ratio of the pixels equal to or larger than the predetermined thresholds in the two wavelength regions is calculated from the fluorescence images obtained by R, G, and B, respectively, but the fluorescence images obtained by G tend to have a high level of benign tumors, and the fluorescence images obtained by B tend to have a high level of malignant tumors. Therefore, in the examples, analysis of Ratio values by G/R for prostates resulted in a range of around 2.0 for benign tumors, 1.7 for healthy subjects, and 1.76 to 1.86 for malignancies, whereas analysis of Ratio values by B/G for colon resulted in a range of 1.9 to 2.0 for malignancies, a range of 0.2 to 0.8 in healthy subjects and around 0.1 in benign tumors.