JP6196159B2 - Metal complex quantum crystals and surface enhanced Raman scattering (SERS) analysis of biochemicals using the same - Google Patents

Description

  TECHNICAL FIELD The present invention relates to a metal complex quantum crystal support having adsorptivity to a biochemical substance as an analyte and having a plasmon enhancing effect, and a biochemical substance analysis using the same, particularly a surface enhanced Raman scattering (SERS) analysis method. .

Surface-modified nanoparticles with nanoclusters formed by controlling the shape and size of metal atoms at the nanometer level are attracting attention as the next generation of typical materials in nanotechnology. This is because new electronic properties are designed by the quantum size effect that will appear in the nanometer range. Here, the “ nanocluster ” is an aggregate formed by collecting several to several hundred atoms / molecules, and its size is several nanometers. These are said to be larger than molecules and smaller than nanocrystals. Nanoclusters are substances that perform unique functions different from atoms, molecules, and bulk solids. Since various functions are exhibited by controlling the size and number of constituent atoms, new knowledge and discoveries on phase transition, crystal growth, chemical reaction, catalysis, etc. are expected. One of them is surface plasmon resonance at the metal surface. In general, electrons in metal do not interact with light, but electrons in metal nanoparticles interact with light under special conditions to cause localized surface plasmon resonance. In particular, in the theoretical consideration of double spheres for silver nanoparticles, it is said that the electric field enhancement around a wavelength of 400 nm is very high at a predetermined inter-particle distance, and below that, a peak exists near the wavelength of 300 nm. Also, the relationship with the particle size is such that the peak position increases as the particle size increases, the peak shifts to the longer wavelength side, and the peak width increases as the particle increases, so it corresponds to a wide range of wavelengths. It is considered that an electric field enhancement effect can be expected.

In the past, research on nanoparticles centered mainly on non-aqueous solvents occupies an overwhelming majority, and very few have steadily studied the crystallization of aqueous nanoparticles. The reason for this is that the aqueous solution is a special solution system in which hydrogen bonds mediate force, and strong hydrogen bonds complicate interparticle forces, and the interaction between metal and ligand is broken by hydrogen bonds of water molecules. There is a report that a quantum crystal was produced in an aqueous solution by using only a bidentate modifier called dicarboxylic acid (Non-patent Document 1). However, in order to realize the creation of various devices using quantum crystals, it is required to create two-dimensional and three-dimensional artificial particle crystals having nanoparticles as constituent elements on a certain carrier . As a result of intensive studies aimed at crystallizing metal quantum dots on a substrate, the present inventors have found that when a metal complex is formed in an aqueous solution, the complex stability constant is high, for example, a multidentate of bidentate or higher. It has been found that a metal complex having a high complex stability constant formed by a ligand can be deposited as a quantum crystal on a metal substrate as a metal complex when deposited by a reduction reaction near its equilibrium potential. In addition, the metal complex can be selected from the ligands that form it, various receptor (receptor) antibodies (for example, human IgE monoclonal antibody), various markers, or biochemical target molecules (for example, analyte) (for example, It has also been found that it has a physical property of adsorbing (CRP protein) and can easily form a solid phase surface suitable for use in various detections (usually standing at 5 ° C. overnight for solid phase immobilization). Furthermore, when the metal of the metal complex is a plasmon metal, it is appropriate to form a quantum crystal (100 to 200 nm) of the metal complex that regularly distributes and encloses quantum dots of nanocluster size (5 to 20 nm). The nano-metal cluster distributed in the region exhibits a localized surface plasmon resonance enhancement effect on Raman light as a metal, and a quantum crystal adsorbs an analyte to form a charge transfer complex, which is suitable for SPR or SERS analysis. Found to form.

On the other hand, studies on gene and protein, provided the opportunity to a new biological marker that can predict the diagnosis of disease (biomarker). In particular, diseases such as cancer can be completely cured through accurate diagnosis at the early stage of onset, and can also prevent recurrence. Therefore, research on the development of diagnostic sensors for early detection of biologically labeled substances by such antigen-antibody reaction is actively progressing. Such sensors can be applied to various fields such as health, environment, military, food, etc. in addition to medical diagnosis, and a very diverse form of research is underway. For detection, there is a method for examining the presence or absence of an antigen using an optical device, but it is the most widely used method to date, despite the disadvantages of time, cost and effort. Recently, a method utilizing the change in the wavelength of light absorbed depending on the presence or absence of an antigen (Non-patent Document 2), through the change of nanoparticles having a surface capable of complementary binding to the antigen depending on the presence or absence of the antigen to be measured. Methods have been reported in which the wavelength of absorbed light is changed (Non-patent Documents 3 and 4 and Patent Document 1).

Among them, in SERS (Surface-Enhanced Raman Scattering, hereinafter SERS) spectroscopy, when molecules are adsorbed on the surface of metal nanostructures such as gold and silver, the intensity of Raman scattering is rapidly increased by 10 ⁶ to 10 ⁸ times or more. It can be developed into a highly sensitive technology that can measure only one molecule directly by combining with nanotechnology that is currently developing at a very fast speed. It has attracted the expected to be used as a.

  Such SERS sensors have a significant technical advantage over electrical nanosensors that change resistance when molecules are adsorbed, because the resistance sensor is a scalar measurement, This is because the SERS sensor obtains the entire spectrum, which is vector amount data, and therefore the amount of information acquired in one measurement is remarkably large.

  For this reason, Kneipp and Nie et al. Reported for the first time that SERS measurements can be performed at a single molecule level on molecules attached to nanoparticles using aggregated nanoparticles. Research on SERS enhancement phenomenon using (nanoparticles, nanoshells, nanowires) was reported. In order to use such a highly sensitive SERS phenomenon for biosensor development, the Mirkin research team recently succeeded in highly sensitive DNA analysis using nanoparticles bound to DNA.

Currently using SERS sensor with a high sensitivity DNA analysis, Me started Alzheimer's disease or diabetes, studies to be performed early diagnosis of various diseases are actively ongoing. That is, the SERS phenomenon provides higher selectivity and higher information than existing measurement techniques such as laser fluorescence analysis in that it directly provides information on the molecular vibrational state or molecular structure provided by Raman spectroscopy. It is recognized as a powerful analytical method for ultrasensitive chemical / biological / biochemical analysis.

Despite these advantages, however, the SERS phenomenon is not only because 1) the mechanism is not fully understood, but 2) the difficulty in synthesizing and controlling nanomaterials that are precisely structurally defined, and 3 ) There are many problems to be solved in terms of reproducibility and reliability due to changes in enhancement efficiency depending on the wavelength of the light used when measuring the spectrum and the polarization direction. It remains as a major issue in the application of the SERS phenomenon.

  Therefore, using the hybrid structure of nanowire and nanoparticle synthesized by vapor phase with precisely defined surface and crystal state, enhancement and measurement of SERS signal of bio-extracts and biomolecules such as protein and DNA A technique for improving reproducibility, sensitivity, and reliability has been proposed (Patent Document 2). Conventional metal-metal nanostructures of this kind provide a 'hot spot' that is an electromagnetic field region that is concentrated by Localized Surface Plasmon Resonance (LSPR) coupling in the nanostructure gaps. Enhanced Raman scattering (SERS) is an attractive technique with high sensitivity for single molecule detection, and SERS provides higher selectivity compared to other detection methods, and Raman spectra are useful for analyte detection. Since it provides a signal for specific chemical groups that can be used, it has been extensively studied as an analytical tool for the detection of various chemicals and the identification of biomolecules such as DNA and proteins since the discovery of this phenomenon.

U.S. Patent No. 6,974,669 JP 2011-81001 A

JSPS Grant-in-Aid for Scientific Research (Fundamental Research (S)) Research Status Report: Keisaku Kimura Chou et al., (2004) Biosens. Bioelectron. 19, 999-1005 Alivisatos et al., (2004) Nat. Biotechnol. 22, 47-52, Namet al., (2003) Science. 301, 1884-1886

  However, the biggest drawback is that metal nanoparticles and a substance to be detected take a long time to form a charge transfer complex necessary for SERS measurement. Accordingly, the present invention provides a plasmon metal substrate on which a substrate and a substance to be detected can easily form a charge transfer complex necessary for SERS measurement in a short time, and using this, a rapid SPR (surface plasmon resonance) of a biochemical substance is provided. ) Or SERS (Surface-Enhanced Raman Scattering) analysis, and the present inventors have conducted extensive research. However, the present inventors have discovered the unique physical properties of the above-described metal complex quantum crystals, and As a result, when a substrate carrying a metal complex quantum crystal is used, it is possible to quickly and easily form a solid-phased surface for immobilizing an antibody, which has been difficult to immobilize in the past. In this case, as a result of performing SPR and SERS analysis methods using the remarkable effect of local plasmon enhancement with respect to Raman light, it was found that the sample has excellent analytical ability. Completed the invention.

In the first invention, a metal complex having a complex stability constant deposited as a metal complex on a metal substrate or metal particle is reduced from the aqueous solution to precipitate a quantum crystal, and the deposited quantum crystal is a biochemical substance ( The metal complex quantum crystal is characterized in that it can adsorb a ligand) or a receptor.

The metal complex is selected so that the complex stability constant represented by the formula (I) correlating with the electrode potential E of the supported metal has 8 or more when represented by the common logarithm (log β) .
Formula (I): E ° = (RT / | Z | F) ln (β)
(Here, E ° is a standard electrode potential, R is a gas constant, T is an absolute temperature, Z is an ion valence, and F is a Faraday constant.)

  When the metal complex is a plasmon metal complex selected from Au, Ag, Pt or Pd, it has a localized surface plasmon resonance enhancing effect on Raman light.

When the metal complex is a silver complex, it is formed by the reaction of the silver complexing agent and silver halide so that the stability constant (production constant) has a logarithm (log β) of 8 or more . Is good.

  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.

On the metal substrate or metal particle, the metal complex crystal that precipitates as a metal complex and an aqueous solution containing the receptor (receptor) is reduced near the equilibrium potential of the metal complex to precipitate the metal complex crystal together with the receptor. When the deposited quantum crystal adsorbs the receptor (receptor) and immobilizes and supports the receptor, it forms a receptor-immobilized metal complex quantum crystal substrate and can adsorb biochemical substances (ligands). A receptor-immobilized substrate can be provided.

  Provided is a receptor-immobilized substrate for SPR or SERS analysis in which the metal complex is a plasmon metal complex and has a localized surface plasmon resonance enhancing effect on Raman light.

  The substrate of the present invention is a metal or metal alloy substrate having a base potential near the equilibrium potential of the metal complex, an aqueous solution containing 500 to 2000 ppm of a plasmon metal complex having a complex stability constant that precipitates the metal complex as quantum crystals, or A metal complex that drops on a particle, starts aggregation on the substrate or particle with a potential difference, removes the metal complex solution from the substrate or particle by gas injection, stops aggregation, and encapsulates the nanometal cluster It can be produced by forming quantum crystals on the substrate or particles.

Accordingly, it dropped by adsorbing liquid containing an analyte biochemical (ligand) on the substrate, and then carrying out more SPR and SERS spectroscopy to measure surface enhanced Raman scattering is irradiated with a laser beam it can.

  A liquid containing a receptor (receptor) is dropped on the SERS substrate to be adsorbed, and then a liquid containing a biochemical substance (ligand) as a specimen is dropped to be adsorbed on the receptor (receptor). SPR and SERS analysis methods can also be carried out by measuring surface-enhanced Raman scattering by irradiating.

In addition, when forming a quantum crystal of a plasmon metal complex, a liquid containing a receptor (receptor) is formed on a SERS substrate in which a liquid containing a biochemical substance (ligand) is mixed in advance with an aqueous plasmon metal complex solution. The SPR and SERS analysis methods can also be carried out by dropping and adsorbing, and then measuring the surface enhanced Raman scattering by irradiating with laser light.

  Furthermore, when forming a quantum crystal of a plasmon metal complex, a biochemical substance (ligand) as an analyte is formed on a SERS substrate in which a liquid containing a receptor (receptor) is mixed in advance with an aqueous plasmon metal complex solution to form a quantum crystal. The SPR and SERS analysis methods can also be carried out by dropping a liquid containing benzene, adsorbing it onto a receptor, and then measuring surface-enhanced Raman scattering by irradiation with laser light.

  The plasmon metal complex quantum crystal of the present invention encloses quantum dots composed of nanoplasmon metal clusters, and the mean diameter of quantum dots composed of nanoclusters has a local surface plasmon resonance enhancing effect on Raman light. It is considered that a complex crystal having a quantum crystal size of 100 to 200 nm adsorbs a biochemical substance (ligand) or a receptor (receptor) to form a charge transfer complex.

  In the present invention, if the stability constant of the plasmon metal complex is 8 or more, it is suitable for producing a quantum crystal of the metal complex. This is because quantum crystals are formed in the form of a metal complex.

In particular, SERS analysis according to the present invention, by using the surface enhanced Raman scattering, there is provided a method for detecting the presence or amount of biochemical substances comprising an analyte, copper or copper from dilute noble metal complex solution Immobilize the receptor on the metal complex quantum crystal deposited on the alloy, bind the analyte to it, and detect biochemical substances from the formed hot spots using surface enhanced Raman scattering spectra Suitable for doing. In particular, a) adding a receptor into an aqueous plasmon metal complex solution to bind the receptor to the plasmon metal complex;
b) preparing a substrate on which a plasmon metal complex solution bonded with a receptor is dropped onto a metal substrate to prepare a plasmon metal complex nanocrystal (quantum crystal) on which a receptor is formed;
c) contacting the analyte on the substrate to bind the analyte to the receptor;
and d) irradiating the quantum crystal in which the substance to be analyzed and the receptor are combined with a laser beam to obtain a surface enhanced Raman scattering spectrum.

  The biochemical substance to be detected includes a cell constituent substance, a genetic substance, a carbon compound, an organic substance that affects the metabolism of the organism, substance synthesis, substance transport or signal transmission process.

  Specifically, the biochemical substances include macromolecular organic substances, organometallic compounds, peptides, carbohydrates, proteins, protein complexes, lipids, metabolites, antigens, antibodies, enzymes, substrates, amino acids, aptamers, sugars, nucleic acids , Nucleic acid fragments, PNA (Peptide Nucleic Acid), cell extracts, or combinations thereof.

  The binding between the quantum crystal receptor and the analyte is characterized as an enzyme-substrate, antigen-antibody, protein-protein, complementary binding between DNA, or biotin-avidin binding. . As specific application methods, a combination of a receptor (receptor) and a biochemical substance (ligand) is an inflammation marker and CRP protein, an undifferentiated marker and an undifferentiated cell, a human IgE monoclonal antibody and an antigen, a tumor marker and an enzyme protein And LAL reagent and endotoxin.

  According to the present invention, the quantum size of the quantum dots contained in the quantum crystal can be controlled by adjusting the aggregation time of the metal complex (the time from dropping to stopping on the metal substrate). A device that demonstrates the effect can be provided. In particular, an element material useful as a surface plasmon resonance excitation element for photoelectric conversion can be provided. The biochemical substance detection method of the present invention does not require pretreatment of the biochemical substance for detection itself, and has a feature capable of directly detecting the biochemical substance itself, and is configured by the quantum crystal according to the present invention. The substrate not only provides an array of metal nanoparticles that form hot spots suitable for plasmon enhancement, but also has a charge to easily immobilize receptors such as antibodies to form charge transfer complexes, making it suitable for SERS detection of proteins Is.

It is explanatory drawing which shows the procedure of the novel SERS board | substrate preparation method shown in Example 1, and the board | substrate made from a limited company Mytec in the upper left is a photograph which shows a right SEM image. 2 is a photograph showing various SEM images of the nanoparticle aggregate (quantum crystal) produced in Example 1. FIG. An enlarged SEM image of the nanoparticles is shown. It is a photograph showing the relationship between the standing time after dropping on the phosphor bronze plate and the quantum crystal shape. It is a graph which shows the result of the EDS spectrum (elemental analysis) of a quantum crystal. It is a graph showing the SERS spectrum of performed rhodamine 6G in Example 2 (10 ^{@ 5} M). Is a graph showing the excitation wavelength dependence of the rhodamine (10 ^{@ 5} M). It is explanatory drawing which shows the application procedure to the antigen antibody reaction detection of the quantum crystal of this invention. It is a graph which shows the detection result of the antigen antibody reaction by the quantum crystal of this invention. It is an electron microscope image of the instrument for endotoxin measurement produced on the surface of a metal plate. The time-dependent change of a Raman scattering spectrum when the standard endotoxin aqueous solution is dripped at the measuring instrument produced combining with the LAL reagent is shown. The peak height in the vicinity of 900 cm −1 of the Raman scattering spectrum measured by dropping an aqueous solution of standard endotoxin into a measuring instrument prepared by reducing the LAL reagent is shown. The peak height in the vicinity of 900 cm −1 of the Raman scattering spectrum measured by dropping aqueous solutions with different concentrations of standard endotoxin into a measuring instrument prepared by reducing the LAL reagent to 1/1000 is shown. 6 is a graph showing a SERS spectrum of a silver thiourea substrate obtained in Example 3. FIG. Congo is a SERS spectrum showing the excitation wavelength dependence of the red (3 × 10 ^{over 6} M).

Example 1
As shown in Fig. 1, a 1,000 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. 2 is a photograph showing various SEM images of the nanoparticle aggregate (quantum crystal) produced in Example 1, and FIG. 3 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. 4 is a photograph showing the relationship between the standing time after dropping on the phosphor bronze plate and the quantum crystal shape. First, it is recognized that a hexagonal quantum crystal is formed and grown while maintaining its shape.
FIG. 5 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.

Consideration of creation of quantum crystals When a quantum crystal is a 1000 ppm silver thiosulfate complex aqueous solution, it is dropped on a phosphor bronze plate and left for 3 minutes to form a hexagonal column shape of around 100 nm. Each hexagonal column-shaped quantum crystal is on the order of several nm. It was confirmed from the SEM images that there were irregularities (FIGS. 1, 2, and 3).
The facets peculiar to metal nanocrystals could not be confirmed, and since elements derived from silver and complex ligands were detected by EDS elemental analysis, the whole was a nanocrystal of a silver complex, and the irregularities appearing on the surface were in the complex It is presumed that silver spreads by forming quantum dots as clusters. When the silver complex quantum crystal of the present invention is formed on a phosphor bronze plate, and the phenomenon that silver-only nanoparticles precipitate on the copper substrate, 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 the phosphor bronze plate , the electrode potential is slightly base, 0.22, so silver It seems that crystals of the complex 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)
Figure 6 shows the results of Rhodamine 6G (10 ^{over 5} M) was dropped on the substrate produced in Example 1, it detected the SERS spectrum with an excitation wavelength of 785nm and 633 nm. The Raman signal enhancement effect by the quantum crystal of the present invention was observed. Figure 7 is a graph showing the excitation wavelength dependence of the rhodamine (10 ^{@ 5} M). In 633 nm excitation, it was the same as a normal resonance Raman spectrum, but in 785 nm excitation, a strong unidentified peak appeared. May be CT resonance effect. However, Red Congo the substrate manufactured in the above Example 1 (10 ^{@ 5} M) was dropped on the substrate produced in Example 1, were detected the SERS spectrum with an excitation wavelength of 785nm and 633nm Congo red ( The peak showing the excitation wavelength dependency of 3 × 10 ⁻⁶ M) was not observed.

(Example 3)
A 1000 ppm aqueous solution of silver thiourea complex in terms of silver was prepared, and one drop was dropped on a phosphor bronze plate, allowed to stand for about 3 minutes, and the solution was blown off to produce a quantum crystal substrate.
FIG. 14 is a graph showing the results of the SERS spectrum of the quantum crystal. Then mixed with Congo Red (3 × 10 ^{over 6} M) and the thiourea 1-one to 1000ppm aqueous solution of silver in terms of silver complex was dropped onto the substrate, and detecting the SERS spectrum with an excitation wavelength of 785nm . FIG. 15 shows the result. Congo red enhanced the Raman signal. For Rhodamine 6G (10 ^{over 5} M) was added and mixed samples was not able to observe the Raman signal enhancement effect.

Or more of the silver thiosulfate complex quantum substrate with thiourea silver quantum crystal substrate from the results is believed that the polarity of the substrate surface are different, Rhodamine 6G is observed in a silver thiosulfate complex quantum substrate, Congo Red (3 × 10 ^{over 6} M) is not observed, rhodamine 6G is not observed in other thiourea silver quantum crystal substrate, Congo Red (3 × 10 ^{over 6} M) results are observed, the former negative polarity, the latter have a positive polarity Presumed to be. By changing the ligand of the metal complex, a metal complex quantum crystal substrate having excellent target molecule adsorptivity can be prepared.

Example 4
The antigen-antibody reaction of the quantum crystal of the present invention was detected by the procedure shown in FIG. An antibody (anti-human IgE monoclonal antibody) is immobilized on a quantum crystal on a substrate formed from a 1000 ppm silver thiosulfate aqueous solution. The human IgE antigen was captured by this, and the SERS spectrum was observed. FIG. 9 is a graph showing the detection result of the antigen-antibody reaction by the quantum crystal of the present invention. A peak peculiar to the antigen-antibody reaction was observed.

(Example 5) Detection of endotoxin Preparation of measuring instrument The concentration of the silver nitrate reagent was 9.7 mM and the concentration of the sodium thiosulfate reagent was 27 mM, and the mixture was stirred and mixed until completely dissolved. The aqueous solution was filtered through a cellulose acetate membrane filter having a pore size of 0.2 μm to remove bacteria and minute insoluble substances. 0.2 mL of the silver nitrate-thiosulfate aqueous solution is put into a glass container containing LAL reagent Limulus-J-Single Test Wako manufactured by Wako Pure Chemical Industries using an autoclave sterilized micropipette (hereinafter referred to as micropipette) and stirred with a vortex mixer. did. 0.06 mL of the solution was dropped onto the surface of a disk-shaped copper alloy plate having a diameter of 7 mm with a micropipette and allowed to stand at room temperature. After 3 minutes, the solution was removed using vaporized gas in a dust-proof spray to produce an endotoxin measuring instrument. FIG. 10 shows an electron microscope image of the measuring instrument attached to the copper alloy substrate. Hexagonal crystalline fine particles not found in the alloy plate are formed by dropping and removing the solution.
2. Change with time 3.4 mL of distilled water for injection (manufactured by Otsuka Pharmaceutical) was dissolved in a vial of standard endotoxin 1700 EU manufactured by Wako Pure Chemical Industries using a micropipette, and then diluted with distilled water for injection to 0.5 EU / mL An endotoxin aqueous solution was prepared. At room temperature, 0.06 mL of the aqueous solution was dropped onto a measuring instrument with a micropipette, attached to a Raman spectrometer, and irradiated with 785 nm laser light to measure a Raman scattering spectrum. Subsequently, Raman scattering spectra were measured after 15 minutes, 30 minutes, 60 minutes, and 90 minutes. As is apparent from the Raman scattering spectrum shown in FIG. 11, a new peak appeared in the vicinity of 900 cm −1 in addition to the main peaks of the measuring instrument 780 cm −1 and 1045 cm −1. Since the intensity of the peak increases with time, it is clear that the peak is due to the endotoxin of the LAL reagent.
3. Amount of LAL reagent used A measurement solution prepared by reducing the LAL reagent used to 1/10, 1/100, and 1/1000 was dropped into a standard endotoxin 5 EU / mL aqueous solution several minutes later, in the same manner as in Example 2. Raman scattering spectrum was measured. FIG. 12 shows the height of the peak in the vicinity of 900 cm @ -1 among the spectral peaks. This is equivalent to the result measured by the measuring instrument before the LAL reagent is reduced by 1/10 or 1/100. In the measuring instrument reduced to 1/1000, the height of the peak is lowered but is about 50% before the reduction, and endotoxin is measured by the measuring instrument prepared with a reagent amount of 1/1000.
4). Measuring instrument with LAL reagent usage reduced to 1/1000 An aqueous solution with a standard endotoxin concentration different from 0.1, 0.5, 5 EU / mL using a measuring instrument prepared by reducing LAL reagent to 1/1000. FIG. 13 shows the height of a peak in the vicinity of 900 cm −1 when the sample was dropped individually on a measuring instrument and the Raman scattering spectrum was measured in the same manner as in Example 2. When the standard endotoxin concentration is 0.1 EU / mL aqueous solution, the height is about 80% compared to 5 EU / mL, and 0.1 EU / mL is measured by the measuring instrument.

Claims (7)

Quantum crystals of a silver complex selected to have a complex stability constant of formula (I) that correlates with the electrode potential E of the supported metal on a metal substrate or metal particle supported metal of copper or copper alloy There,
Formula (I): E ° = (RT / | Z | F) ln (β)
(Here, E ° is a standard electrode potential, R is a gas constant, T is an absolute temperature, Z is an ion valence, and F is a Faraday constant.)
Is 8 or more when the stability constant of the silver complex represented by common logarithm (Logbeta), metal and having a quantum crystal structure is capable adsorbing biochemical substances (ligands) or receptor (receptor) complex quantum crystals. A method of reducing a metal complex from an aqueous solution on a metal substrate or a metal supported on metal particles to precipitate quantum crystals,
While selecting a copper or copper alloy as the supported metal, in selecting a silver complex as the metal complex, it is selected to have a complex stability constant represented by formula (I) that correlates with the electrode potential E of the supported metal,
Formula (I): E ° = (RT / | Z | F) ln (β)
(Here, E ° is a standard electrode potential, R is a gas constant, T is an absolute temperature, Z is an ion valence, and F is a Faraday constant.)
A metal complex quantum characterized in that the silver complex is formed by a reaction of a silver complexing agent and silver halide so that the stability constant (generation constant) is 8 or more when represented by a common logarithm (log β). Crystal production method. In the silver halide silver chloride, the complexing agent is a thiosulfate, thiocyanate, sulfite, thiourea, potassium iodide, thiosalicylic acid salts, according to claim 2, wherein the one selected from thiocyanuric acid salt A method for producing a metal complex quantum crystal. A liquid containing a biochemical substance (ligand) as an analyte is dropped and adsorbed onto a support on which a quantum crystal of the silver complex according to claim 1 is formed, and then laser-irradiated to perform surface enhanced Raman scattering. SPR and SERS analysis methods characterized by measuring. A liquid containing a receptor (receptor) is dropped and adsorbed onto a support on which quantum crystals of the silver complex according to claim 1 are formed, and then a liquid containing a biochemical substance (ligand) as an analyte is dropped. SPR and SERS analysis method characterized by measuring surface-enhanced Raman scattering by adsorbing to a receptor and then irradiating a laser beam. When forming a carrier in which quantum crystals of the silver complex according to claim 1 are formed , an analyte is formed on the carrier in which a liquid containing a receptor (receptor) is mixed in advance with an aqueous silver complex solution to form quantum crystals. A SPR and SERS analysis method characterized in that a liquid containing a biochemical substance (ligand) is dropped and adsorbed on a receptor (receptor), and then laser-irradiated to measure surface enhanced Raman scattering. When forming a carrier in which quantum crystals of the silver complex according to claim 1 are formed, a solution containing a biochemical substance (ligand) is mixed in advance with an aqueous silver complex solution, and the receptor is formed on the carrier in which quantum crystals are formed. A method of SPR and SERS analysis, characterized in that a liquid containing a body (receptor) is dropped and adsorbed and then irradiated with a laser beam to measure surface enhanced Raman scattering.