JP2006030155A - Plate assay using spectroscopy - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a plate assay method that uses spectroscopy. <P>SOLUTION: (1) A compound to be measured is fixed and arranged on a substrate surface, and the compound, having a functional group for forming bonding in specific reaction with the object to be measured allows a metal particle having plasmon absorption by adsorption or bonding to the surface to operate on the compound to be measured, energy is applied to the surface, and the energy of the plasmon absorption is measured, thus performing the detection and quantitative analysis of the compound to be measured. (2) The functional group that forms bonding in specific reaction with a compound to be measured in which the metal particle, where the compound to be measured is adsorbed and bonded to the particle surface is fixed and arranged on the substrate surface is allowed to operate on metal particles having plasmon absorption, energy is applied to the substrate, and the energy of the plasmon absorption is measured, thus performing the detection and quantitative analysis of the compound to be measured. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to an analysis method using spectroscopy. More specifically, an object to be measured or a compound having a functional group that specifically binds to the object to be measured is fixedly arranged on the surface of the substrate, and the object to be measured or the object to be measured is fixedly arranged on the surface of the substrate. The compound having a functional group that specifically binds to the surface is allowed to act on metal particles having plasmon absorption, which are modified on the surface. After bonding the substrate and the particles, the unreacted compound is removed by washing and remains. The present invention relates to an analytical method for measuring the type and / or amount of a compound to be measured by measuring plasmon absorption. The present invention can be applied to the analysis of various compounds, but can be usefully applied to the field of biotechnology in which high sensitivity and high speed are particularly required in recent years.

  Decoding the base sequence of 3 billion human genomes has been completed, and the post-genomic era has come. With the completion of the human genome decoding, we moved to the era of proteomic research to investigate the cause of disease by comprehensively examining the expression of genetic information at the protein level as post-genomic research. Proteome research has resulted in the identification of proteins that cause disease. By elucidating proteins that cause diseases and their behaviors, it becomes possible to diagnose and treat diseases aimed at tailor-made drug discovery and protein expression. The study of protein-nucleic acid interactions aimed at the foregoing is rapidly expanding.

  Moreover, the combination exists astronomically in analyzing the influence of drugs between and on biomolecules. For this reason, a high-sensitivity and high-speed analysis method and apparatus are required. The present invention provides a method for detecting and / or analyzing various biological materials and interactions specific to biological materials in large quantities and simultaneously in a highly sensitive and high speed manner.

  Examples of compounds to be measured include nucleic acids, proteins, sugars, cells, antibodies, antigens, enzymes, receptors and environmental hormones. In recent years, when the influence of environmental hormones on the living body is regarded as a problem, the demand for analysis in this field has been increasing rapidly. Specific examples of environmental hormones include compounds described in Non-Patent Document 1.

  As means for performing high-throughput analysis in the field of biotechnology, molecular arrays called DNA chips, protein chips, sugar chain chips, and cell chips are widely used. A molecular array generally immobilizes a known probe (functional group or compound that specifically acts on a compound to be measured) such as nucleic acid or protein on a substrate surface, or immobilizes an analyte on a substrate, A sample solution or probe is dispensed on the array and allowed to act for a certain period of time, and unreacted sample is removed by washing as necessary. The bond (state) expressed by the specific interaction is detected by an optical or electrochemical method. From the signal intensity and pattern information obtained by the measurement, it is possible to perform qualitative and / or quantitative analysis of the measurement object and / or quantitative analysis of the interaction strength between the measurement compound and the probe.

The molecular array as a DNA chip is intended to determine a DNA sequence (Non-Patent Document 2, Non-Patent Document 3) or detect a mutation in a specific DNA sample (Non-Patent Document 4).
Hereinafter, the shape of the support for the molecular array, the method for immobilizing the probe or the compound to be measured, and the method for detection disclosed in the prior art will be described in detail.

  As the shape of the support, a flat plate type, a bead type, a capillary type, a fiber type, a μ-TAS type and the like are currently commercially available.

  Representative probes include nucleic acids, peptides, sugars and the like. There are two types of probe immobilization methods. The first is a method of sequentially synthesizing and immobilizing probes in the form of a support (Patent Documents 1 and 2), and the second is a method of fixing a separately prepared probe to a support (Patents). Reference 3). As an apparatus for immobilizing the probe on the support of the second method, a pin, an ink jet, a stamp or the like is applied.

As a method for detecting the interaction, it is common to label the compound or probe to be measured with a fluorescent dye and to detect the luminescence of the spot due to the interaction spectroscopically. In this case, a charge coupled device (CCD) is generally used as the detector. In addition, a plate reader, a scanner, or a digital camera commercially available from Perkin Elmer can be used as appropriate according to the required accuracy. In addition, there are the following methods for measuring by a physical method without labeling. Electrochemical techniques that detect the conductivity that changes with the interaction, surface plasmon resonance (SPR) sensors that measure changes in the dielectric constant that change due to adsorption or binding, and interactions on the quartz crystal Quartz crystal microbalance (QCM) for detecting changes in the vibration frequency to be expressed, ellipsometry for obtaining the thickness increment of the thin film due to the interaction by polarized reflected light on the surface, as in the case of polarization analysis Atomic Force Microscopy (AFM), where the change in thickness is due to interaction and the surface is scanned with a needle to measure the increment in thickness. In addition, a classical immunological technique using an antibody that recognizes an interaction is also used. In some cases, a mass analysis system is also used as a detector, and structural analysis of the collected compound is also performed.

  In recent years, DNA arrays that have been widely used are used for analysis of DNA hybridization phenomena, sequence analysis (Non-patent Document 5), and detection of mutation sites. In the molecular array represented by a DNA array, it is generally labeled with a fluorescent dye or an electrochemically active compound, a sample is reacted, and qualitative and / or quantitative analysis is performed depending on the presence and intensity of fluorescence. However, when labeling with a fluorescent dye, it is difficult to control the site and number to which the labeling agent binds, and the physiological and chemical properties change due to the labeling. Moreover, when it has a long sequence, an analysis is difficult (nonpatent literature 4).

  Surface plasmon resonance (SPR) and crystal oscillator (QCM) detect interactions by measuring changes in dielectric constant and mass change caused by biological substances bound to biomolecules immobilized on gold thin films. High sensitivity and no labeling required. Specifically, it is used for analysis of the strength of protein-nucleic acid interaction and qualitative / quantitative analysis. However, commercially available “BIACORE” (Biacore AB, Uppsala, Sweden) and Nippon Laser Corp. SPR devices cannot obtain information from only changes in the dielectric constant of the metal surface due to adsorption / bonding. And non-specific adsorption by contaminants. Also, SPR is not suitable for high-throughput analysis in which many samples are analyzed at a high speed at a time because of its mechanical structural characteristics.

  Enzyme immunoassay is widely used mainly in the field of medical examination and quarantine, and the method has already been established. In particular, in recent years, it has also been used in the field of environmental measurement for the purpose of BSE (bovine spongiform encephalopathy) testing, pesticide measurement, and detection of specific bacteria, and the number of tests using this technique has increased rapidly. However, an operation process of operation is required, and actual measurement requires a large dedicated device and skill of a skilled expert. In addition, the enzyme reaction has a problem that it is greatly affected by various external factors, particularly the reaction temperature. The long time required for analysis is also a big problem for mass inspection.

Yasushi Iguchi, CM Publishing, Latest Trends in Environmental Hormones and Measurement / Test / Equipment Development (2003)

Mirzabekov, Trends in Biotechnology 12, 27-32, (1994)

Pease et al. Proc. Natl. Acd. Sci. USA 91, 5022-5026 (1994)

Winzeler et al. Science 281, 1194-1197 (1998).

JP 2004-141152 A

JP 2001-517300 A

Japanese Patent No. 3272365

Mirzabekov, Trends in Biotechnology, 12, 27-32, 1994

Automation Technology for Genome Characterisation, Wiley-Interscience (1997), T.W. J. et al. Begelsdijk, 205-225

  In view of the above, an object of the present invention is to provide a means for quantitatively and / or qualitatively analyzing multiple samples with high sensitivity and high speed.

  The material used for the plate is preferably a material having high light transmittance when measuring the transmitted light, and examples thereof include single crystal calcium fluoride, silicon, ceramics, glass, metal, and polymer. Specific examples of the polymer material include polytetrafluoroethylene, polypropylene, polystyrene, and polycarbonate.

  The compound that specifically forms a bond with the compound to be measured must be kept fixed on the substrate surface without being washed away in the reaction process and in the cleaning process for cleaning unreacted compounds. If necessary, a linker may be interposed between the object to be measured and / or the compound that specifically forms a bond with the compound to be measured and the substrate and the metal particles. As specific linkers, compounds described in catalogs of Dojindo, Sigma, and Piece can be used.

  Nucleic acid and the like can be fixed and arranged on the calcium fluoride substrate by treating the substrate surface with an aminosilane-based polymer, and the thiol group in the protein is firmly adsorbed on the gold surface without interposing a linker, It can be stably fixed.

  Any substrate can be used, but flat plates, well structures, beads, capillaries, fibers, and the like that can measure multiple specimens at high speed can be used. Since the amount of immobilization of the compound depends on the surface area, the surface of the substrate is preferably smooth and small in variation.

  The reagent may be supplied to the substrate surface in any of a solid state and / or a solution state and / or a gas state. In the case of providing in a solution state, a chip type dispenser such as System Biotics PlatePrep 400 or Genex Sampling System manufactured by Kaken Genex can be used. It is also possible to use an ink jet apparatus capable of precisely controlling the discharge amount. Specific examples of the inkjet dispensing apparatus include Stampman manufactured by Nippon Laser Electronics, MRMJ manufactured by Mect Dispenser, and the like, and can be accurately and evenly dispensed onto the substrate surface.

In measurement, the sample is preferably wet with an aqueous solution. In particular, in a biological sample, the three-dimensional structure differs between a wet state and a dry state, and the affinity between the compound to be measured and the probe may change. Further, since the plasmon absorption energy of the metal particles having plasmon absorption varies depending on the dielectric constant in the vicinity, the plasmon absorption spectrum is different between the dry state and the wet state. That is, the plasmon absorption spectrum varies due to variations depending on the drying rate. Since it is easier to completely moisten than to control the drying rate, it is preferable to perform measurement after completely moistening.
The metal particles having plasmon absorption include at least one metal selected from the group consisting of gold, silver, copper, platinum, aluminum, zinc, tin, rhodium, palladium, or iridium, nickel ruthenium, osmium, and iridium. Is included. Gold and silver colloidal aqueous solutions are commercially available from Tanaka Kikinzoku, British Biocell, Polyscience, and the like. In the case of metal particles composed of two or more kinds of metals, either a core-shell structure or a solid solution structure may be used. The metal particle diameter is preferably in the range of 1 nm to 250 nm, and more preferably in the range of 5 nm to 100 nm. Metal particles having a particle size larger than 250 nm may settle due to gravity, which necessitates stirring of the reaction solution, and is inappropriate for high throughput intended for multi-analyte processing. On the other hand, when the particle diameter is smaller than 1 nm, it is difficult to introduce a large amount of ligand on the particle surface.

  It is preferable that the metal particles are not aggregated before formation of a bond by a specific reaction between the compound to be measured and the probe. In the aggregated state, the specific reaction may be slowed and / or inhibited, which is inappropriate for high throughput. Adsorption of a polymer such as polyethylene glycol and / or a polyethylene glycol derivative on the surface of the metal particles is preferable for the following two reasons. 1) The dispersion is stabilized by the entropy effect of the polymer. It can be stably stored in a solution having an ionic strength or higher at a biological level (H. Otsuka, Y. Akiyama, Y. Nagasaki, K. Kataoka: J. Am. Chem. Soc., 123, 8226 ( 2001) and T. Ishii, H. Otsuka, Y. Nagasaki, K. Kataoka: Langmuir, 20, 561-564 (2004)). Commercially available colloidal gold is generally reduced with citric acid, and citrate ions or acetone dicarboxylic acid, which is an oxidant of citric acid, is adsorbed on the surface. And / or dispersed by electrostatic repulsion by acetone dicarboxylic acid. Metal particles dispersed and stabilized only by electrostatic repulsion force are electrostatically shielded and agglomerate in an environment with high ionic strength. 2) It is known that non-specific adsorption due to proteins and the like can be suppressed by adsorbing hydrophilic polymers, particularly highly biocompatible polyethylene glycol or polyethylene glycol derivatives, on the surface of metal particles ( Y. Mori, S. Nagaoka, H. Takiuchi, T. Kikuchi, N. Noguchi, H. Tanzawa, Y. Noshiki: Trans. Am. Soc. Artif. When protein is adsorbed on the hydrophilic polymer, the interaction between the polymer and water increases and the enthalpy increases, the degree of freedom of the three-dimensional structure due to adsorption, and the entropy decreases due to the increase in osmotic pressure. Thermodynamically difficult to place. In addition, protein adsorption changes the environment around polyethylene glycol to hydrophobic, and the activation energy that changes polyethylene glycol from a highly polar structure to a less polar structure becomes very high, resulting in nonspecific adsorption of proteins. It is suppressed. Metal particles, particularly gold and silver particles, bind to thiols in biomolecules, and nonspecific adsorption tends to occur. For coating metal particles with a polyethylene glycol derivative, see Y. et al. Mori, S.M. Nagaoka, H .; Takiuchi, T .; Kikuchi, N .; Noguchi, H .; Tanzawa, Y .; Noishiki: Trans. Am. Soc. Artif. Internal. Organs, 28, 459 (1982) and T.W. Ishii, H.I. Otsuka, Y. et al. Nagasaki, K .; It can be implemented by the method disclosed in Kataoka: Langmuir, 20, 561-564 (2004).

Visible light, ultraviolet light, infrared light, or near infrared light can be used as the irradiated light. Metal particles made of gold, silver or the like having a particle diameter of 250 nm or less have light absorption based on plasmon vibration of electrons in the visible light region. Specifically, the absorption wavelength of the gold fine particles is, for example, S.I. Linkand, M.M. A. ElSaedJ. Phys. Chem. B, 103, 4212-4217 (1999). For silver fine particles, see N.C. Lepold, B.M. Lendl, J.M. Phys. Chem. B, 107, 5723-5727 (2003) and A.I. Henglein, M.M. Giersig, J.M. Phys. Chem. B, 103, 9533-9539 (1999). According to the aspect of the present invention, metal particles having plasmon absorption are bound to either the compound to be measured and / or the compound having a functional group that specifically reacts with the compound to be measured to form a bond. When a bond is formed by a specific reaction, the compound to be measured that has formed a specific reaction can be discriminated and analyzed by removing unreacted substances. It is also possible to measure using surface enhanced Raman spectroscopy as disclosed in Yukihiro Ozaki, Tamtake Ito, Hyundai Kagaku, September, 20-27 (2003). Many biological samples emit fluorescence in a wavelength range of 650 nm to 800 nm. This wavelength region overlaps with the Raman shift when excited with short-wavelength excitation light, and further increases the quantum efficiency of fluorescence generation when excited with short-wavelength excitation light. Will be shielded. For this reason, when measuring using a Raman scattered light, it is preferable to irradiate near infrared light, More preferably, it is an area | region of 800 nm-1600 nm.
The solvent is preferably an aqueous solution containing 50 wt% or more of water, and more preferably 90% or more. A solvent having a low water content, that is, containing a large amount of an organic compound may dissolve and / or deform the substrate. In addition, when biological components are to be measured, protein denaturation with an organic solvent may occur. It is not suitable because it may cause If necessary, the solvent can have a buffering capacity, and a commercially available buffering agent can be added in an appropriate amount.
When the concentration of RNA or DNA to be measured is low, it may be amplified by PCR transcription or cloning if necessary.

The analysis method according to the present invention is a simple and highly sensitive analysis method in which non-specific adsorption is suppressed. The analysis method can be suitably used for high-throughput biomolecule analysis.

  Examples and the like specifically showing the configuration and effects of the present invention will be described below.

Example 1
[Preparation of gold colloid modified with biotin antibody on its surface (hereinafter referred to as biotin antibody-gold colloid)]
A gold colloid whose surface was modified with a biotin antibody (hereinafter referred to as biotin antibody-gold colloid) was prepared by the following procedure. To 1 ml of a colloidal gold solution (particle size: 40 nm, 0.01 wt / vol%, manufactured by Polysciences), 150 ul of a 0.4 μmol / l biotin antibody aqueous solution was added and mixed by inverting at room temperature for 10 minutes. To this solution, 100 μl of a 10 mM sodium phosphate buffer solution (pH 7.4, 0.15 M NaCl, hereinafter referred to as sodium phosphate buffer solution) in 1.5 mg / ml MeO-PEG-SH (molecular weight 10,000) was added, and the mixture was mixed with 1 at room temperature. After mixing by inversion for an hour, centrifugation (4 ° C., 4000 G, 45 minutes) was performed, and the sediment was dispersed in a sodium phosphate buffer containing 0.1 wt / vol% BSA. The absorbance at 520 nm of the obtained biotin antibody-gold colloid solution was measured, and sodium phosphate buffer containing 0.1 wt / vol% BSA was used so as to match the absorbance at 520 nm of the unmodified gold colloid solution. The concentration was adjusted.

[Preparation of biotinylated BSA]
Biotinylated BSA was prepared by the following procedure. Dissolve 1 mg of sodium phosphate buffer in 5 mg of BSA and add DMSO solution of 20 mg / ml Biotin- (AC5) 2-Osu (manufactured by Dojindo) so that the molar ratio of BSA and Biotin is 1:10 And mixed by inversion for 2 hours at room temperature. This was gel-filtered with PD10 to remove unreacted Biotin- (AC5) 2-Osu. About the obtained biotinylated BSA, the amount of biotin introduced was measured by the HABA method. As a result, it was found that 7 molecules of biotin were bonded to 1 molecule of BSA.

[Preparation of biotinylated plate]
The biotinylation was immobilized on the plate surface by the following procedure to prepare a biotinylated BSA plate. The biotinylated BSA prepared by the above method was dissolved in a sodium phosphate buffer solution to prepare a 2 μg / ml to 7.8 ng / ml biotinylated BSA sodium phosphate aqueous solution. 50 μl each of the above-mentioned concentrations of biotinylated BSA sodium phosphate buffer solution and sodium phosphate buffer solution are dispensed into microplates (Nunc Immunoplate, MaxiSorp, manufactured by Nargenunk), and allowed to stand at 4 ° C. overnight. The biotinylated BSA was modified on the plate. The biotinylated BSA solution is discarded, the plate is washed with a sodium phosphate buffer, and excess biotin or BSA not fixedly arranged on the surface of the plate is washed away. Then, 1 wt / vol% MeO-PEG / PLA ( Blocking was performed by dispensing 100 ul of sodium phosphate buffer solution (containing 10 vol / vol% MeOH) of PEG part molecular weight 5000 and polylactide part molecular weight 5000) at room temperature for 1 hour. The plate was washed with a sodium phosphate buffer containing 0.05 wt / vol% Tween 20, and MeO-PEG / PLA not fixedly arranged on the surface was washed away to prepare a biotin plate.
50 μl of biotin antibody-gold colloid solution was dispensed onto a biotinylated plate and allowed to stand overnight at 4 ° C., followed by shaking at room temperature for 1 hour. The biotin antibody-gold particles that did not bind to the biotinylated plate surface were washed and washed away with a sodium phosphate buffer containing 0.05 wt / vol% Tween 20, and then immediately absorbed with a plate reader (520 nm, 0.1 second, 25 points / well).

Comparative Example 1
As a result of the same operation as in Example 1 except that water was used instead of the 0.4 μmol / l biotin antibody aqueous solution in the preparation of gold colloid, regardless of the concentration of the biotinylated BSA solution used for modification, No change in absorbance at 520 nm was observed.

Example 2
[Preparation of biotin antibody-gold colloid]
To 1 ml of a colloidal gold solution (particle size: 40 nm, 0.01 wt / vol%, manufactured by Polysciences), 150 ul of a 0.4 μmol / l biotin antibody aqueous solution was added and mixed by inverting at room temperature for 10 minutes. To this solution was added 100 μl of 1.5 mg / ml MeO-PEG-SH (molecular weight 10000) sodium phosphate buffer solution, and the mixture was mixed by inverting at room temperature for 1 hour, followed by centrifugation (4 ° C., 4000 G, 45 minutes). And the precipitate was dispersed in a sodium phosphate buffer containing 0.1 wt / vol% BSA. The absorbance of the obtained biotin antibody-gold colloid solution was measured at 520 nm, and a sodium phosphate buffer containing 0.1 wt / vol% BSA so that the absorbance was doubling with respect to 520 nm of the unmodified gold colloid solution. The concentration was adjusted with the liquid. This was further diluted 2 ⁿ with a sodium phosphate buffer containing 0.1 wt / vol% BSA from 1 to 32 times to prepare biotin antibody-gold colloids having six concentrations.

[Create biotin plate]
Distribute 50 μl of 0.5 μg / ml biotinylated BSA sodium phosphate buffer solution to a microplate (Nunc Immunoplate, MaxiSorp, manufactured by Nargenunk), and allow to stand overnight at 4 ° C. Modified to plate. After discarding the biotinylated BSA solution and washing the plate with sodium phosphate buffer, 100 ul of 1 wt / vol% MeO-PEG / PLA sodium phosphate buffer solution (containing 10 vol / vol% MeOH) is added to each well. The solution was dispensed and blocked by shaking at room temperature for 1 hour. The plate was washed with a sodium phosphate buffer containing 0.05 wt / vol% Tween 20 to prepare a biotin plate.

50 μl each of biotin antibody-gold colloid solutions having different concentrations were dispensed onto a biotin plate, left at 4 ° C. overnight, and then shaken at room temperature for 1 hour. The biotin antibody-gold particles that did not bind to the biotinylated plate were washed and washed away with a sodium phosphate buffer containing 0.05 wt / vol% Tween 20, and then immediately absorbed with a plate reader (520 nm, 0.1 sec, 25 Point / well) was measured.

Comparative Example 2
As a result of the same operation as in Example 2 except that 0.4 μmol / l rabbit antibody was used in place of the 0.4 μmol / l biotin antibody aqueous solution in preparation of the gold colloid, regardless of the concentration of the gold colloid solution. No change in absorbance at 520 nm was observed.

Example 3
[Preparation of biotin antibody-gold colloid]
To 1 ml of a colloidal gold solution (particle size: 40 nm, 0.01 wt / vol%, manufactured by Polysciences), 150 ul of a 0.4 μmol / l biotin antibody aqueous solution was added and mixed by inverting at room temperature for 10 minutes. To this solution was added 100 μl of 1.5 mg / ml MeO-PEG-SH (molecular weight 10000) sodium phosphate buffer solution, and the mixture was mixed by inverting at room temperature for 1 hour, followed by centrifugation (4 ° C., 4000 G, 45 minutes). And the precipitate was dispersed in a sodium phosphate buffer containing 0.1 wt / vol% BSA. The absorbance at 520 nm of the obtained biotin antibody-gold colloid solution was measured, and sodium phosphate buffer containing 0.1 wt / vol% BSA was used so as to match the absorbance at 520 nm of the unmodified gold colloid solution. The concentration was adjusted.

[Create biotin plate]
A sodium phosphate buffer solution of 2 μg / ml biotinylated BSA was diluted 2 ⁿ with sodium phosphate buffer from 1-fold (2 μg / ml) to 256-fold (7.8 ng / ml). 50 μl each of biotinylated BSA sodium phosphate buffer solution and sodium phosphate buffer solution at each concentration was dispensed into a microplate (Nunc Immunoplate, MaxiSorp, manufactured by Nargenunk), and allowed to stand at 4 ° C. overnight. Biotinylated BSA was modified on the plate. The biotinylated BSA that was not fixedly arranged on the plate surface was washed and washed away with sodium phosphate buffer, and then 1 wt / vol% MeO-PEG / PLA in sodium phosphate buffer solution (10 wt / vol% MeOH) was added to each well. Were dispensed in 100 ul aliquots and blocked by shaking at room temperature for 1 hour. The plate was washed with a sodium phosphate buffer containing 0.05 wt / vol% Tween 20 to prepare a biotin plate.

50 μl of a biotin antibody-gold colloid solution was dispensed onto a biotin plate and allowed to stand overnight at 4 ° C., followed by shaking at room temperature for 1 hour. The biotin antibody-gold particles that did not bind to the biotinylated plate surface were washed and washed away with a sodium phosphate buffer containing 0.05 wt / vol% Tween 20, and then immediately absorbed with a plate reader (520 nm, 0.1 second, 25 points / well).

Comparative Example 3
The preparation of colloidal gold was carried out in the same manner as in Example 3 except that 0.4 μmol / l rabbit antibody was used instead of the 0.4 μmol / l biotin antibody aqueous solution. As a result, the biotinylated BSA solution used for modification was used. No change in absorbance at 520 nm was observed regardless of the concentration of (Comparative Example 3-1). Separately, instead of 0.4 μmol / l biotin antibody aqueous solution, 1 μmol / l BSA was used, and instead of 100 μl of 1.5 mg / ml MeO-PEG-SH (molecular weight 10,000) sodium phosphate buffer solution, 1.5 mg / Ml MeO-PEG-SH (molecular weight 5000) was used in the same manner as in Example 3 except that 50 μl of sodium phosphate buffer solution was used. As a result, regardless of the concentration of the biotinylated BSA solution used for modification, A change in absorbance at 520 nm was not confirmed (Comparative Example 3-2).

Example 4
[Preparation of biotin antibody-gold colloid]
To 1 ml of a colloidal gold solution (particle size: 40 nm, 0.01 wt / vol%, manufactured by Polysciences), 150 ul of a 0.4 μmol / l biotin antibody aqueous solution was added and mixed by inverting at room temperature for 10 minutes. To this solution was added 1.5 mg / ml MeO-PEG-SH (molecular weight 10,000) sodium phosphate buffer solution (100 μl of solution, and mixed by inverting at room temperature for 1 hour, followed by centrifugation (4 ° C., 4000 G, 45 minutes). The precipitate was dispersed in a sodium phosphate buffer solution containing 0.1 wt / vol% BSA, and the absorbance of the obtained biotin antibody-gold colloid solution at 520 nm was measured, and 520 nm of the unmodified colloidal gold solution was measured. The biotin antibody-gold colloid was prepared by adjusting the concentration with a sodium phosphate buffer containing 0.1 wt / vol% BSA so as to coincide with the absorbance at.

[Create biotin plate]
50 μl of 0.5 μg / ml biotinylated BSA sodium phosphate buffer solution was dispensed into a microplate and allowed to stand overnight at 4 ° C. to modify the biotinylated BSA on the plate. The biotinylated BSA solution that was not fixedly arranged on the plate surface was washed and washed away with a sodium phosphate buffer solution, and then 1 wt / vol% MeO-PEG / PLA in a sodium phosphate buffer solution (10 wt / wt) was added to each well. 100 ul each) (containing vol% MeOH) was shaken at room temperature for 1 hour for blocking. The plate was washed with a sodium phosphate buffer containing 0.05 wt / vol% Tween 20 to prepare a biotin plate.

50 μl of a biotin antibody-gold colloid solution was dispensed onto a biotin plate and allowed to stand overnight at 4 ° C., followed by shaking at room temperature for 1 hour. The biotin antibody-gold particles that did not bind to the biotinylated plate surface were washed and washed away with a sodium phosphate buffer containing 0.05 wt / vol% Tween 20, and then immediately absorbed with a plate reader (520 nm, 0.1 second, 25 points / well).

Comparative Example 4
Instead of 1 wt / vol% MeO-PEG / PLA in sodium phosphate buffer solution (including 10 wt / vol% MeOH) in the preparation of biotin plates, 1 wt / vol% gelatin / sodium phosphate buffer solution was used. The same operation as in Example 4 was performed except that blocking was performed.

  Diagram showing the reactivity of colloidal gold modified with biotin antibody (biotin antibody-gold colloid) and colloidal gold not modified with biotin antibody to biotinylated plates

  Diagram showing the correlation between biotin antibody-gold colloid concentration and the amount bound to the plate, and that the rabbit antibody does not bind to the biotinylated plate

  Diagram showing the reactivity of biotin antibody-modified colloidal gold (biotin antibody-gold colloid) and rabbit antibody or BSA-modified gold colloid to biotinylated plates

  Figure comparing the blocking effect of gelatin and MeO-PEG / PLA

Claims (15)

  A compound to be measured is fixedly arranged on the surface of the substrate, and metal particles having plasmon absorption formed by adsorption and / or bonding to the surface of a compound having a functional group that reacts specifically with the compound to be measured to form a bond are covered. A method of detecting and / or quantitatively analyzing a compound to be measured by acting on a measurement compound, irradiating the substrate with energy, and measuring the energy of plasmon absorption.   A metal group formed by binding and / or adsorbing a compound to be measured to a metal particle having plasmon absorption reacts specifically with the compound to be measured which is fixedly arranged on the surface of the substrate to act on a functional group that forms a bond. A method for detecting and / or quantitatively analyzing a compound to be measured by irradiating the substrate with energy and measuring the energy of plasmon absorption.   3. The analysis according to claim 1, wherein the object to be measured is at least one selected from the group consisting of nucleic acids, proteins, sugars, cells, antibodies, antigens, enzymes, receptors and environmental hormones. Method.   The metal particles having plasmon absorption are at least one or more selected from the group consisting of gold, silver, copper, platinum, aluminum, zinc, tin, rhodium, palladium, or iridium, nickel, ruthenium, osmium, and iridium. The analysis method according to claim 1, further comprising a metal.   5. The metal particle according to claim 4, wherein a particle diameter of the metal particle having plasmon absorption is in a range of 1 to 100 nm.   The analysis method according to claim 1, wherein the energy applied to the substrate is at least one selected from the group consisting of ultraviolet light, visible light, infrared light, and near infrared light.   The analysis method according to claim 1, wherein a blocking agent that suppresses nonspecific adsorption is adsorbed and / or bound to the surface of the substrate and / or the metal particles.   3. The analysis method according to claim 1, wherein the blocking agent that suppresses nonspecific adsorption on the surface of the substrate and / or the metal particles is alkylene glycol and / or an alkylene glycol derivative.   The analysis method according to claim 1, wherein the blocking agent that suppresses nonspecific adsorption is polyethylene glycol and / or a polyethylene glycol derivative.   The polyethylene glycol derivative according to claim 9, wherein the polyethylene glycol derivative is a block copolymer of polyethylene glycol and polylactide.   11. The polyethylene glycol derivative according to claim 9, wherein the polyethylene glycol and polylactide block copolymer has a polyethylene glycol part molecular weight of 500 to 50,000 and a polylactide part molecular weight of 500 to 50,000. .   The compound to be measured and / or the compound having a functional group that specifically reacts with the compound to be measured to form a bond is immobilized on the substrate surface and / or the metal particle surface through a linker. The analysis method according to claim 2 of claim 1.   The compound to be measured and / or the compound having a functional group that specifically reacts with the compound to be measured to form a bond are directly fixed to the substrate surface and / or the metal particle surface without a linker. The analysis method according to claim 1 of claim 1.   3. The analysis method according to claim 1, wherein the material used for the substrate is at least one selected from the group consisting of single crystal calcium fluoride, silicon, ceramics, glass, metal, and polymer. .   3. The analysis method according to claim 1, wherein the detector is a digital image reader.

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