WO2014181814A1 - 生体試料のラマン定量分析用バイオチップ - Google Patents
生体試料のラマン定量分析用バイオチップ Download PDFInfo
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- WO2014181814A1 WO2014181814A1 PCT/JP2014/062315 JP2014062315W WO2014181814A1 WO 2014181814 A1 WO2014181814 A1 WO 2014181814A1 JP 2014062315 W JP2014062315 W JP 2014062315W WO 2014181814 A1 WO2014181814 A1 WO 2014181814A1
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- 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
- G01N21/65—Raman scattering
- G01N21/658—Raman scattering enhancement Raman, e.g. surface plasmons
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- C—CHEMISTRY; METALLURGY
- C01—INORGANIC CHEMISTRY
- C01G—COMPOUNDS CONTAINING METALS NOT COVERED BY SUBCLASSES C01D OR C01F
- C01G5/00—Compounds of silver
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- 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
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54366—Apparatus specially adapted for solid-phase testing
- G01N33/54373—Apparatus specially adapted for solid-phase testing involving physiochemical end-point determination, e.g. wave-guides, FETS, gratings
- G01N33/5438—Electrodes
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- 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
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/551—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being inorganic
- G01N33/553—Metal or metal coated
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- 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
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
- G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
- G01N33/57496—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites involving intracellular compounds
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- C—CHEMISTRY; METALLURGY
- C01—INORGANIC CHEMISTRY
- C01P—INDEXING SCHEME RELATING TO STRUCTURAL AND PHYSICAL ASPECTS OF SOLID INORGANIC COMPOUNDS
- C01P2002/00—Crystal-structural characteristics
- C01P2002/60—Compounds characterised by their crystallite size
Definitions
- the present invention relates to a biochip for Raman quantitative analysis of biological samples.
- microarray analysis often plays a major role in regulating protein function, but cannot detect, identify or quantify protein modifications.
- biosensors have recently received a lot of attention due to their increasing use in chemical, biological and pharmaceutical research and disease diagnosis.
- Various biomolecules consisting of high recognition elements and conversion structures that convert molecular recognition events into quantifiable signals, including oligonucleotide pairs, antibody-antigen, hormone-receptor, enzyme-substrate and lectin-glycoprotein interactions Although it has been developed to detect complexes, it is still insufficient.
- the wavelength of the Raman emission spectrum is characteristic of the chemical composition and structure of the Raman scattering molecules in the sample, and the intensity of the Raman scattered light depends on the concentration of the molecules in the sample.
- the SERS phenomenon is also used when 1) the mechanism is not fully understood, 2) the difficulty of synthesizing and controlling nanomaterials that are precisely structurally defined, and 3) the spectrum.
- the present invention provides a biochip for SERS analysis in which a protein in each sample is easily adsorbed and fixed on a chip, and DNA can be easily quantified by laser irradiation, and a specific protein can be easily quantified.
- An object of the present invention is to provide a method capable of analyzing disease identification and progression from a specific protein profile including DNA.
- the present inventors have found that when forming a metal complex in an aqueous solution, the complex stability constant is high, for example, a high complex stability constant formed with a multidentate ligand of bidentate coordination or higher. It has been found that when a metal complex having a metal complex is precipitated by a reduction reaction near its equilibrium potential, the metal complex can be deposited as a quantum crystal on the metal substrate. Further, the present inventors have found that a metal complex exhibits physical properties for adsorbing proteins in a biological sample and easily forms a solid-phased surface suitable for use in various detections.
- the metal of the metal complex is a plasmon metal
- the distributed nano metal cluster exhibits surface plasmon resonance enhancement effect on Raman light as a metal, and the quantum crystal adsorbs the analyte to form a charge transfer complex to form a biochip suitable for SPR or SERS analysis. I found out.
- the present invention allows specific amounts of protein, including DNA in plasma, to be quantified by dropping plasma onto a metal complex quantum crystal, and also includes DNA between normal and cancer patients.
- DNA DNA in plasma
- a biological sample selected from the group consisting of urine, blood, plasma, serum, saliva, semen, stool, sputum, cerebrospinal fluid, tears, mucus, exhaled components, etc.
- a Raman spectrum is obtained by irradiating a protein in a biological sample immobilized on a crystal with a laser beam of a specific wavelength, and a comprehensive spectrum such as the peak height, peak integration value, and peak expression time of the obtained Raman spectrum is used.
- a metal complex aqueous solution containing a plasmon metal complex selected from Au, Ag, Pt or Pd is dropped onto a supported metal having an electrode potential lower than that of the complex metal, and the metal complex is formed on the support metal. Crystallized as nano-sized quantum crystals.
- the metal complex is selected to have a complex stability constant (log ⁇ ) equal to or higher than the complex stability constant (log ⁇ ) represented by the formula (I) correlated with the electrode potential E of the supported metal.
- E ° (RT /
- the surface property or polarity is preferably adjusted in the aqueous solution before or after crystallization of the metal complex quantum crystal on the supported metal according to the detection target in the biological sample.
- the antigen or antibody can be dispersed in the quantum crystal in the same manner as the ligand when the antigen or antibody is mixed with the metal complex aqueous solution before crystallization to precipitate the quantum crystal.
- the antigen or antibody mixed in the metal complex aqueous solution seems to be mixed and deposited at the time of precipitation of the metal complex as well as the complex ligand. Therefore, it can be used for detection using plasma protein binding, detection using calcium binding protein, detection using sugar binding protein (lectin) (infection, immune disease). It can be used to detect various diseases (viruses such as Alzheimer, HIV, influenza).
- silver peroxide is affected by the electrode potential of the substrate in the case of silver thiosulfate quantum crystals by alkali treatment using an aqueous solution of sodium hypochlorite as an alkaline aqueous solution containing halogen ions. It was found that the mesocrystals, which are superstructures arranged in a three-dimensional array in the form of neurons, were recrystallized by aggregation of the contained silver oxide by self-organization.
- the metal complex is a silver complex
- it is formed by the reaction of a silver complexing agent having a stability constant (generation constant) (log ⁇ i ) of 8 or more and silver halide, and the complexing agent includes thiosulfate, If it is one kind selected from thiocyanate, sulfite, thiourea, potassium iodide, thiosalicylate, and thiocyanurate, not only silver ions are reduced, but they can be precipitated as quantum crystals of silver complexes. Has been found.
- the silver complex has quantum dots composed of nanoclusters having an average diameter of 5 to 20 nm, and the size of the quantum crystal is 100 to 200 nm.
- the silver concentration in the aqueous solution of the metal complex is preferably 500 to 2000 ppm.
- the quantum crystal is silver thiosulfate
- the silver oxide containing silver peroxide obtained by alkali treatment sodium hypochlorite aqueous solution
- halogen ions is assembled by self-assembly.
- the mesocrystals which are superstructures arranged three-dimensionally in the form of neurons, exhibit not only their structural features but also negative charges in water, and can form positive charge-related cancer-related substances to form charge-transfer complexes.
- silver particles can be precipitated by light irradiation, and that there is a region where surface plasmon enhancement effect can be obtained by laser irradiation. Therefore, according to the present invention, a biochip for quantifying a cancer-related substance Raman can be provided.
- the biochip according to the present invention is suitable for adsorption of a sample that tends to be negatively charged in an aqueous solution because it tends to be positively charged due to the characteristics of the metal complex.
- the ligand can be mixed into the quantum crystal by mixing an appropriate ligand in the metal complex aqueous solution.
- endotoxin can be detected by precipitating it on a substrate by mixing a Limulus reagent (LAL reagent) in the quantum crystal together with the aqueous metal complex solution of the present invention.
- a detection reagent such as an antibody can be solid-phased using the properties of the quantum crystal deposited on the substrate.
- the biochip according to the present invention can be recrystallized as a metal oxide crystal by subjecting a metal complex quantum crystal to an alkali treatment in the presence of a halogen ion.
- Silver oxide mesocrystals containing silver peroxide (AgO or Ag 2 O 3 ) are formed by alkali treatment of silver thiosulfate quantum crystals in the presence of chloride ions. Quantify specific proteins, including DNA in biological fluids, predict various diseases, and know their progress.
- biological sample means a sample containing an analyte containing several hundred proteins such as a biological fluid of a host. Samples may be directly subjected to Raman analysis or may be pretreated to denature or fragment protein-containing molecules in the sample so that they can be detected more easily.
- the analyte of interest is a target analyte such as a specific binding pair member complementary to the analyte of interest that is detected only when the analyte of interest is present in the sample.
- a substance that demonstrates the analyte is the analyte that is detected in the assay.
- the biological sample may be, for example, urine, blood, plasma, serum, saliva, semen, stool, sputum, cerebrospinal fluid, tears, mucus, and exhaled components.
- protein includes peptides, polypeptides and proteins and protein-containing analytes such as antigens, glycoproteins, lipoproteins and the like.
- a method for obtaining protein composition information from a complex biological sample, such as a patient sample.
- Proteins in biological samples can be dissolved and diluted in aqueous solutions or hydrophilic solvents.
- the protein in the sample may be denatured using agents selected from reducing agents, surfactants, chaotropic salts, and the like.
- agents selected from reducing agents, surfactants, chaotropic salts, and the like.
- Common chemicals that can be used to reduce disulfide bonds include, but are not limited to, DTT, DTE, 2-mercaptoethanol, and the like.
- Representative surfactants that can be used to denature proteins include sodium dodecyl sulfate (SDS), lithium dodecyl sulfate (LDS), Triton X 100 (registered trademark), Tween-20 (registered trademark), etc. But is not limited thereto.
- Typical chaotropic salts that can be used to denature proteins include, but are not limited to, GuSCN, NaSCN, GuClO 4 , NaClO 4 and urea.
- Solid proteins such as slices can be denatured using chemical cleaving agents to digest the protein or serine-proteases such as trypsin.
- the protein may be in a native structure (native) for Raman spectroscopy or SERS analysis.
- the metal complex quantum crystal of the present invention is deposited on a metal to form a biochip, the metal nanodod, which functions as nanometal particles, is easily metallized and contacts with a sample (target molecule) in an aqueous solution. It appears to form a transfer complex. Therefore, it combines the surface plasmon resonance enhancement effect of regularly arranged metal nanoparticles and the ionic metal property that easily forms a charge transfer complex with the target molecule (we combine the metal property and the ionic property, and the metal and metal ion Therefore, the biochip suitable for surface enhanced Raman scattering (SERS) measurement can be provided.
- SERS surface enhanced Raman scattering
- the metal complex is selected so as to have a complex stability constant (log ⁇ ) equal to or greater than the formula (I) that correlates with the electrode potential E of the supported metal.
- a complex stability constant (log ⁇ ) equal to or greater than the formula (I) that correlates with the electrode potential E of the supported metal.
- E ° (RT /
- R is a gas constant
- T is an absolute temperature
- Z is an ion valence
- F is a Faraday constant.
- the metal complex is a plasmon metal complex selected from Au, Ag, Pt or Pd, it has a surface plasmon resonance enhancing effect on Raman light.
- the metal complex is a silver complex, it is preferably formed by a reaction between a silver complexing agent having a stability constant (generation constant) (log ⁇ i ) of 8 or more and silver halide.
- the silver halide is preferably silver chloride, 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 concentration of the metal complex in the aqueous solution should be determined mainly in consideration of the size of the quantum crystal to be formed, and when using a dispersant, the concentration should also be considered. Usually, it can be used in the range of 100 ppm to 5000 ppm, but depending on the function of the ligand, a concentration of 500 to 2000 ppm is preferred to prepare a nanosize that should be called a nanocluster.
- Quantum crystals formed on a metal substrate or metal particles are likely to have a positive polarity in an aqueous solution as a metal complex crystal.
- an alkaline solution such as a pH of 11 or more is used. It is preferable to adjust the polarity by dripping sodium hypochlorite aqueous solution. Metal oxide mesocrystals formed by alkali treatment tend to exhibit negative polarity in an aqueous solution. Immobilization of the protein in the sample to the biochip crystal can be promoted.
- Proteins generated due to diseases are contained in biological samples such as urine, blood, plasma, serum, saliva, semen, stool, sputum, cerebrospinal fluid, tears, mucus, and exhalation components.
- concentration of the dropped sample is dissolved in an aqueous solution or a hydrophilic solvent and diluted to an appropriate concentration.
- FIG. 3 is a plasma sample of a colon cancer patient, which is a Raman spectrum measured with a 633 nm laser (30 mW) diluted 10 times, 100 times, 500 times, 1000 times and 10,000 times with pure water.
- the peak rise value (PSV) and the peak integral value change with the concentration. Therefore, it can be seen that quantitative analysis of specific proteins including DNA in plasma can be performed.
- FIG. 1 shows a method for calculating a peak of a Raman waveform, and it is confirmed that the spectrum of Raman scattering by a 633 nm laser of a human plasma sample forms peaks of scattering intensity around 1350 cm ⁇ 1 and 1550 cm ⁇ 1 . Therefore, the maximum increase value (pm) based on the average value (m) of the scattering intensities at 800 cm ⁇ 1 (a) and 2000 cm ⁇ 1 (b) was defined as the peak increase value (Shifting Peak Value: PSV). . Moreover, the area of the whole peak was defined as a peak integrated value.
- These peak rise values and peak integration values are important for viewing the amount of a specific protein including DNA in human plasma, and can be used as an index showing the identification and progression of cancer along with the peak onset time.
- the method for calculating the peak of the Raman waveform is shown, and the spectrum of Raman scattering by a 633 nm laser of a human serum sample shows that peaks of scattering intensity are formed around 1350 cm ⁇ 1 and 1550 cm ⁇ 1 . It is a Raman spectrum figure of the sample which adjusted the serum obtained from 12 gastric cancer patients. It is a Raman spectrum figure of the sample which adjusted the serum obtained from 12 colorectal cancer patients. It is a Raman spectrum figure of the sample which adjusted the serum obtained from 12 benign disease patients. It is a graph which shows the comparison of the Raman scattering peak rise value of stomach cancer, colon cancer, and a benign disease sample.
- 2 is a photograph showing various SEM images of the nanoparticle aggregate (quantum crystal) produced in Example 1.
- the XPS measurement result of the alkali-treated recrystallized substrate is shown.
- the XPS measurement result after etching the surface of a recrystallized substrate is shown.
- Example 1 As shown in Fig. 4, a 1000 ppm aqueous solution of silver thiosulfate was prepared, one drop was dropped on a phosphor bronze plate, left for about 3 minutes, and the solution was blown away to produce a quantum crystal showing an SEM image on the right side. It had been.
- FIG. 5 is a photograph showing various SEM images of the nanoparticle aggregate (quantum crystal) produced in Example 1, and FIG. 6 shows an enlarged SEM image of the nanoparticles. It is a thin hexagonal columnar crystal of about 100 nm, and the surface has irregularities on the order of several nm. No facets specific to metal nanocrystals could be confirmed.
- FIG. 7 is a photograph showing the relationship between the standing time after dropping on the phosphor bronze hill and the quantum crystal shape.
- FIG. 8 is a graph showing the results of EDS spectrum (elemental analysis) of the quantum crystal.
- the crystals formed on the phosphor bronze plate detected elements derived from silver and complex ligands, but prepared a 1000 ppm aqueous solution of silver thiosulfate on the copper plate, dropped one drop and left it for about 3 minutes. When it was blown away, only silver was detected.
- the equilibrium potential of the silver thiosulfate complex is 0.33 and the copper Since it is equivalent to the electrode potential (0.34), only silver (0.80) is deposited on the copper substrate, and in the case of phosphor bronze, the electrode potential is slightly base, 0.22, so that the silver complex It is probable that the crystals were precipitated. Therefore, in order to prepare a quantum crystal, 1) the complex aqueous solution is a dilute region of 500 to 2000 ppm, and 2) the electrode potential of the supported metal is slightly lower than the equilibrium potential of the metal complex aqueous solution. ) It seems to be important that the metal complex is aggregated by the electrode potential difference. The same was true when a 1000 ppm thiourea silver complex aqueous solution was used.
- Example 2 A sodium hypochlorite aqueous solution of pH 11 was dropped on the silver thiosulfate quantum crystal substrate on the phosphor bronze plate prepared in Example 1, and after 3 minutes, the aqueous solution was blown off. Immediately thereafter, serum obtained from 12 gastric cancer patients was collected. Pure, 10-fold diluted sample, serum obtained from 12 colorectal cancer patients, pure 10-fold diluted sample, and serum obtained from 12 benign disease patients, pure 10-fold adjustment Each of the obtained samples was irradiated with a laser beam of 633 nm, and a Raman spectrum was measured. It can be said that there is a correlation between the degree of progression of gastric cancer and colorectal cancer and the peak rise value and peak integral value.
- FIG. 2D is a graph showing a comparison of the Raman scattering peak increase values of gastric cancer, colon cancer, and benign disease samples. It can be seen that the peaks of gastric cancer samples and colon cancer samples are significantly higher for patients with benign diseases. It can be said that it is difficult to find the difference in the peak rise value between the gastric cancer sample and the colorectal cancer sample, but considering the peak onset time and the peak integral value, it is possible to identify both cancers. it can.
- the target free DNA to be detected is wound around a protein called histone, which is equivalent to a bobbin, and the unit structure (one set) is called a nucleosome, and the structure in which nucleosomes are gathered and formed into a string is called chromatin ( Fiber).
- histone protein
- the unit structure one set
- nucleosome the structure in which nucleosomes are gathered and formed into a string
- chromatin Fiber
- methylation that makes DNA difficult to unravel easily, but usually histones are charged (+), DNA is charged (-), and two are like magnets They are stuck together and cannot be dissolved by methylation, and the DNA wound around the histone is charged (+) (see FIG. 11 (a)).
- the peak near 529 eV is an O peak derived from silver peroxide (AgO)
- the peak near 530 eV is silver oxide (Ag 2 O). It is recognized as an O peak derived from it.
- the amount of oxygen decreases, but the O peak derived from silver peroxide (AgO) in the peak near 529 eV is larger than the O peak derived from silver oxide (Ag2O) in the peak near 530 eV. It can be said that silver peroxide is formed in the vicinity of the substrate. This is presumed to be due to the catalytic action during mesocrystal formation and the electrode potential of the substrate.
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Abstract
Description
式(I):E゜= (RT/|Z|F)ln(βi)
(ここでE゜は、標準電極電位、Rは、気体定数、Tは、絶対温度、Zは、イオン価、Fは、ファラデー定数を表す。)。
式(I):E゜ = (RT/|Z|F)ln(βi)
(ここでE゜は、標準電極電位、Rは、気体定数、Tは、絶対温度、Zは、イオン価、Fは、ファラデー定数を表す。)
金属錯体が、Au、Ag、PtまたはPdから選ばれるプラズモン金属の錯体である場合は、ラマン光に対して表面プラズモン共鳴増強効果を有する。
金属錯体が銀錯体であるときは、安定度定数(生成定数)(log βi)が8以上の銀錯化剤とハロゲン化銀との反応により形成されるのがよい。ハロゲン化銀としては塩化銀が好ましく、錯化剤としてはチオ硫酸塩、チオシアン酸塩、亜硫酸塩、チオ尿素、ヨウ化カリ、チオサリチル酸塩、チオシアヌル酸塩から選ばれる1種であるのが好ましい。
(実施例1)
図4に示すように、チオ硫酸銀1000ppm水溶液を調製し、その1滴をりん青銅板上に滴下し、約3分間放置し、溶液を吹き飛ばすと、右横のSEM像を示す量子結晶が作成されていた。
図5は実施例1で製造したナノ粒子凝集体(量子結晶)の各種SEM像を示す写真であり、図6はナノ粒子の拡大SEM像を示す。100nm前後の薄い六角柱状結晶であって、表面に数nmオーダの凹凸が発現している。金属ナノ結晶に特有のファセットは確認できなかった。
図7はりん青銅坂上に滴下後の放置時間と量子結晶形状の関係を示す写真である。まず、六角形の量子結晶が生成し、形状を維持しつつ成長するのが認められる。
図8は量子結晶のEDSスペクトル(元素分析)の結果を示すグラフである。りん青銅板上に形成された結晶は銀及び錯体配位子由来の元素を検出したが、銅板上にチオ硫酸銀1000ppm水溶液を調製し、その1滴を滴下し、約3分間放置し、溶液を吹き飛ばした場合は、銀のみを検出したに過ぎなかった。
量子結晶は1000ppmチオ硫酸銀錯体水溶液の場合、りん青銅板上に滴下して3分間放置すると、100nm前後の六角柱状に形成され、各六角柱状の量子結晶は数nmオーダの凹凸を持つことがSEM像から確認された(図4、図5及び図6)が、金属ナノ結晶に特有のファセットは確認できず、EDS元素分析で銀及び錯体配位子由来の元素を検出されたため、全体は銀錯体のナノ結晶であって、その表面に現れる凹凸は錯体中の銀がクラスタとして量子ドットを形成して広がっていると推測される。本発明の銀錯体量子結晶がりん青銅板上に形成される一方、銅基板上には銀のみのナノ粒子が析出する現象を見ると、チオ硫酸銀錯体の平衡電位が0.33で銅の電極電位(0.34)と同等であるため、銅基板上には銀(0.80)のみが析出し、りん青銅の場合は0.22と電極電位がわずかに卑であるため、銀錯体の結晶が析出したものと思われる。したがって、量子結晶を作成するためには1)錯体水溶液が500~2000ppmという希薄な領域であること、2)金属錯体水溶液の平衡電位に対し担持金属の電極電位がわずかに卑であること、3)電極電位差で金属錯体が凝集させることが重要であると思われる。また、1000ppmチオ尿素銀錯体水溶液を使用した場合も同様であった。
実施例1で調整したりん青銅板上のチオ硫酸銀量子結晶基板にpH11の次亜塩素酸ナトリウム水溶液を滴下し、3分後水溶液を吹き飛ばし、その直後、胃癌患者12例から得られた血清を純粋で10倍希釈して調整した試料、大腸がん患者12例から得られた血清を純粋で10倍希釈して調整した試料および良性疾患患者12例から得られた血清を純粋で10倍調整した試料のそれぞれを633nmのレーザー光を照射してラマンスペクトルを測定した。胃がんおよび大腸がんの進行度とピーク上昇値およびピーク積分値との間には相関関係が認められるということができる。また、胃がんの場合、ラマンスペクトルはレーザー照射後1分後に、大腸がんの場合はレーザー照射後2~3分後にラマンスペクトルにピークが発現した。また、図2Dは胃癌、大腸がん、良性疾患試料のラマン散乱ピーク上昇値の比較を示すグラフである。良性疾患患者に対し、胃癌試料および大腸がん試料のピークは有意に高いことが認められる。胃癌試料と大腸がん試料とはピーク上昇値では差を見つけるのが困難であるということができるが、ピーク発現時間およびピーク積分値を考慮すると、両者のがん同定は可能であるということができる。ここで、検出すべき対象の遊離DNAは糸巻きに相当するヒストンというタンパク質に巻き付いており、ひと巻きされた単位構造(1セット)はヌクレオソームと呼び、ヌクレオソームが集まりひも状になった構造をクロマチン(線維)と呼ぶ。そして、細胞ががん化して分裂を繰り返すとき、がんが増えるのに都合の悪い遺伝子(がん抑制遺伝子)が出てこないようしっかりヒストンに巻きついて蓋をし、ヒストンへの巻き方をさらにきつくして、DNAが簡単にはほどけないようにして、メチル化という修飾が起こっているが、通常ヒストンは(+)、DNAは(-)にチャージされていて、2つは磁石のようにくっつきあい、しかもメチル化して解けないようになっており、ヒストンに巻き付いたDNAは(+)に帯電している(図11(a)参照)。他方、アセチル化は(-)にチャージするため、通常は(+)のヒストンがアセチル化されれば、(-)同士となってDNAと反発する。すると、DNAという‘糸’がヒストンからほどけて遺伝子が発現するメカニズムとなっている(図11(b)参照)。したがって、癌細胞由来の遊離DNAを選択的に吸着させるには、ヒストンに巻き付いたDNAは(+)に帯電しているので、吸着させる基板は(-)に帯電しているのが好ましいと考えられる。
上記量子結晶基板に5%次亜塩素酸ソーダ水溶液を滴下して2分間処理して除去すると図12に示す結晶構造が見られ、針状の結晶とラクビーボール状の塊と大きい塊が見られたので、それぞれの組成をEDSスペクトル(元素分析)で分析すると、以下の反応式から針状の結晶はともに塩化銀と酸化銀の複合結晶からなるものと考えられるが、図12の結果は塩素は確認できず、銀と酸素が支配的であることがわかる。
Na2S2O3+4NaClO+H2O →Na2SO4+H2SO4+4NaCl (1)
Ag+ + NaCl → AgCl + Na+ (2)
Ag+ + 3NaOCl → 2AgCl + NaClO3 + 2Na+ (3)
Ag+ + OH- → AgOH (4)
2Ag++ 2OH → Ag2O +H2O (5)
したがって、本発明に係るメソ結晶の形成には銀イオンとチオ硫酸イオンが塩素イオンの存在下にアルカリ酸化反応により生ずるものと思われるが、通常の水溶液中では酸化銀が形成されるに過ぎないが、以下のXPS測定から過酸化銀が支配的に形成されていると推測される。
XPS測定:
上記量子結晶基板に次亜塩素酸ナトリウム水溶液25μlを2分間滴下し、再結晶基板を作り、エッチングせずそのまま(使用機種: アルバック・ファイ(株)/PHI5000 Versa Probe II(走査型X線光電子分光分析装置))でAgとOとをXPS測定した。また、比較対象のため、酸化銀の粉と塩化銀の粉のAgを測定した。他方、再結晶基板をアルゴンガスクラスターイオン銃で5分間エッチングしてAgとOをXPS測定した。図13及び図14のXPS測定結果を図12に基づくEDSの結果から推測すると、529eV付近のピークは過酸化銀(AgO)に由来するOピークで、530eV付近のピークは酸化銀(Ag2O)に由来するOピークであると認められる。エッチングした場合に、酸素量は減少するが、529eV付近のピークの過酸化銀(AgO)に由来するOピークが、530eV付近のピークは酸化銀(Ag2O)に由来するOピークよりも大きいことは基板近傍に過酸化銀が形成されているのを物語るものといえる。これは、メソ結晶形成時の触媒作用と基板の電極電位が影響しているものと推測される。
なお、EDS測定は上記再結晶基板を使用機種: 日本電子株式会社/JSM-7001F(電界放出形分析走査電子顕微鏡)を用いて行った。
また、チオ硫酸銀の量子結晶を次亜塩素酸水溶液、0.01規定苛性ソーダ水溶液、0.01規定塩酸水溶液、0.1モル炭酸ナトリウム水溶液で処理しても同様の結果は得られなかった。よって、この針状結晶の形成には銀イオンとチオ硫酸イオンの存在下に上記反応により生ずるものと思われる。酸化銀は水溶液中で負電荷を帯び、光により還元されて金属銀を析出させる。過酸化銀はその傾向が顕著なので、正電荷の癌関連物質を吸着し、しかも吸着した癌関連物質と銀粒子との間の表面プラズモン増強効果が得られるものと思われる。
Claims (5)
- Au、Ag、PtまたはPdから選ばれるプラズモン金属の錯体を含む金属錯体水溶液を錯体金属より卑なる電極電位を有する担時金属上に滴下し、担体金属上で金属錯体をナノサイズの量子結晶として晶出させてなり、金属錯体は担持金属の電極電位Eと相関する式(I)で示される錯体安定度定数(logβ)以上を有するように選択され、
式(I):E゜= (RT/|Z|F)ln(βi)
(ここでE゜は、標準電極電位、Rは、気体定数、Tは、絶対温度、Zは、イオン価、Fは、ファラデー定数を表す。)
担持金属上の金属錯体量子結晶が晶出前の水溶液中または晶出後に検出対象に応じて表面性状を調節してなることを特徴とするプラズモン金属錯体の量子結晶からなるバイオチップ。 - 担持金属上の金属錯体量子結晶が晶出前の水溶液中または晶出後に検出対象に応じて表面性状または電荷を調節してなる請求項1記載のバイオチップ。
- 金属錯体が銀錯体であるときは、安定度定数(生成定数)(log βi)が8以上の銀錯化剤とハロゲン化銀との反応により形成され、錯化剤としてはチオ硫酸塩、チオシアン酸塩、亜硫酸塩、チオ尿素、ヨウ化カリ、チオサリチル酸塩、チオシアヌル酸塩から選ばれる1種である請求項1記載のバイオチップ。
- 銀錯体は平均直径が5~20nmであるナノクラスタからなる量子ドットを有し、量子結晶のサイズが100~200nmとなる請求項1記載のバイオチップ。
- 請求項4の銀錯体量子結晶をハロゲンイオンの存在下でアルカリ処理して得られる過酸化銀を含む銀酸化物ナノ結晶(メソ結晶)を含むラマン定量用バイオチップ。
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JPWO2015170711A1 (ja) * | 2014-05-08 | 2017-04-20 | 有限会社マイテック | プラズモニックチップおよびそれを用いるがん疾病の蛍光画像ならびにラマン分光による診断方法 |
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US10215700B2 (en) | 2015-02-26 | 2019-02-26 | Mytech Co., Ltd. | Plasmonic chip for observing cancer related substances by localized surface plasmon resonace |
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