WO2006022277A1 - 表面プラズモン共鳴分析における解離定数の算出方法 - Google Patents
表面プラズモン共鳴分析における解離定数の算出方法 Download PDFInfo
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- WO2006022277A1 WO2006022277A1 PCT/JP2005/015318 JP2005015318W WO2006022277A1 WO 2006022277 A1 WO2006022277 A1 WO 2006022277A1 JP 2005015318 W JP2005015318 W JP 2005015318W WO 2006022277 A1 WO2006022277 A1 WO 2006022277A1
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- surface plasmon
- plasmon resonance
- liquid
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- molecule
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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
- G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
- G01N21/55—Specular reflectivity
- G01N21/552—Attenuated total reflection
- G01N21/553—Attenuated total reflection and using surface plasmons
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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
- G01N33/536—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase
- G01N33/542—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase with steric inhibition or signal modification, e.g. fluorescent quenching
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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
Definitions
- the present invention relates to a method for calculating a dissociation constant between a molecule interacting with an analyte molecule immobilized on a metal surface in surface plasmon resonance analysis.
- SPR surface plasmon resonance
- QCM quartz crystal microbalance
- SPR measurement technology detects the adsorption and desorption near the surface by measuring the refractive index change in the vicinity of the organic functional film in contact with the metal film of the chip by measuring the peak shift of the reflected light wavelength or the change in the amount of reflected light at a certain wavelength.
- QCM measurement technology is a technology that can detect the adsorption / desorption mass at the ng level from the change in the oscillation frequency of the oscillator due to the adsorption / desorption of the substance on the gold electrode (device) of the crystal oscillator.
- QCM measurement technology is a technology that can detect the adsorption / desorption mass at the ng level from the change in the oscillation frequency of the oscillator due to the adsorption / desorption of the substance on the gold electrode (device) of the crystal oscillator.
- SPR surface plasmon resonance
- a commonly used measurement chip is composed of a transparent substrate (eg, glass), a deposited metal film, and a thin film having a functional group capable of immobilizing a physiologically active substance thereon, via the functional group. Immobilize a physiologically active substance on the metal surface. The interaction between biomolecules is analyzed by measuring a specific binding reaction between the physiologically active substance and the analyte substance.
- Examples of the surface plasmon resonance measuring apparatus for performing the analysis as described above include an apparatus described in JP-A-2001-330560.
- Analyte binding to (metal film + ligand) is detected as refractive index change (and accompanying dark line angle change).
- the so-called “sensor gram” can be used to obtain changes over time of the signal (such as the amount of binding). It is important to fit a velocity equation such as the following equation (i) to the sensorgram and then obtain velocity coefficients such as the adsorption velocity coefficient (ka) and the separation velocity coefficient (kd).
- kd Adsorption rate coefficient
- kd Desorption rate coefficient
- C Analyte concentration knowledge
- Rmax Theoretical maximum binding amount
- t Time
- the liquid in the flow path system as described above does not contain the test substance to be measured!
- the physiological activity can be obtained.
- the noise width of the signal change of the reference cell and the baseline fluctuation within the measurement time become problems, and reliability It was difficult to obtain high binding detection data.
- Japanese Patent Application No. 2003-404040 describes the flow path system including cells formed on a metal film and the intensity of the light beam totally reflected on the metal film surface.
- a surface plasmon resonance detecting device for detecting a surface plasmon resonance state
- a measurement method characterized by measuring a change in surface plasmon resonance in a state of being allowed to enter is described. After changing the liquid in the flow path system in this way, by measuring the change in surface plasmon resonance with the liquid flow stopped, the noise level (noise width of the reference chip) is low and the baseline fluctuation ( It is possible to obtain highly reliable measurement results with a small signal change of the reference chip.
- the dissociation constant between a molecule interacting with an analyte molecule immobilized on a metal surface in surface plasmon resonance is calculated with a low noise level (reference chip noise width) and baseline fluctuation ( It was an issue to be solved by realizing an analysis method that can obtain measurement results with small signal change (reference chip signal) and high reliability.
- the present inventors have also obtained a method for measuring a signal change of surface plasmon resonance in a state where the flow of the liquid is stopped after exchanging the liquid in the flow path system in the surface plasmon resonance measuring apparatus. It was demonstrated for the first time that the rate constants such as kd (adsorption rate coefficient) and kd (desorption rate coefficient) could be obtained accurately, and the present invention was completed.
- the flow path system including cells formed on the metal film and the light for detecting the surface plasmon resonance state by measuring the intensity of the light beam totally reflected on the metal film surface
- a method of measuring a signal change of surface plasmon resonance using a surface plasmon resonance measuring apparatus comprising a detecting means, after replacing the liquid in the flow path system, the liquid flow is stopped. From the measurement result of the change, a method for calculating the dissociation constant between the molecule immobilized on the metal surface and the interacting molecule is provided.
- ka is the adsorption rate coefficient
- kd is the desorption rate coefficient
- c is the concentration of the analyte molecule near the metal surface.
- X is the distance of the metal surface force
- D is the diffusion coefficient of the molecule to be analyzed
- c is the concentration of the molecule to be analyzed.
- R represents the surface plasmon signal
- Rmax represents the signal when the analyte molecule is saturated and adsorbed.
- the dissociation constant is calculated by correcting the time taken to exchange the liquid as an adsorption phenomenon under ideal conditions.
- the dissociation constant is calculated using nonlinear regression analysis.
- the dielectric block Preferably, the dielectric block, a metal film formed on one surface of the dielectric block, a light source that generates a light beam, and the dielectric block with respect to the dielectric block;
- An optical system that allows the total reflection condition to be obtained at the interface with the metal film and that includes various incident angle components, a flow path system that includes cells formed on the metal film, and the interface.
- a surface plasmon resonance measuring apparatus is used which comprises a light detection means for detecting the state of surface plasmon resonance by measuring the intensity of the totally reflected light beam.
- the liquid in the flow path system does not contain the test substance to be measured !, and is exchanged from the control liquid to the sample liquid containing the test substance to be measured.
- the surface plasmon resonance change is measured with the flow stopped.
- a reference cell that does not bind a substance that interacts with a test substance and a detection cell that binds a substance that interacts with the test substance are connected in series and installed in a flow path system, A liquid is allowed to flow through the reference cell and the detection cell.
- the ratio (Ve / Vs) of the liquid exchange amount (Ve ml) per measurement to the volume (Vs ml) of the cell (Ve / Vs) is not less than 100 and not more than 100. More preferably, Ve / Vs is 1 or more and 50 or less.
- the time required for exchanging the liquid in the channel system is 0.01 seconds or more and 100 seconds or less.
- the present invention relates to a method for calculating a dissociation constant between a molecule that interacts with an analyte molecule immobilized on a metal surface. More specifically, the method for calculating the dissociation constant according to the present invention includes a flow path system including cells formed on a metal film, and the intensity of a light beam totally reflected on the metal film surface to measure the surface.
- a surface plasmon resonance measuring device comprising a light detection means for detecting a state of plasmon resonance, and after changing the liquid in the flow path system, the signal change of the surface plasmon resonance is stopped in a state where the flow of the liquid is stopped
- the dissociation constant of the molecule interacting with the analyte molecule immobilized on the metal surface is calculated from the signal change measurement result.
- the state of surface plasmon resonance is detected by measuring the intensity of a flow path system including cells formed on a metal film and the intensity of a light beam totally reflected on the metal film surface.
- a surface plasmon resonance measuring apparatus comprising a light detecting means for measuring the signal change of the surface plasmon resonance in a state where the flow of the liquid is stopped after the liquid in the flow path system is exchanged.
- a method for measuring the signal change of the surface plasmon resonance described above which includes calculating the dissociation constant between the molecule immobilized on the metal surface and the interacting molecule from the measurement result of the signal change.
- the molecule to be analyzed adsorbs over time to a molecule that interacts with the molecule to be analyzed immobilized on the metal surface. This phenomenon can be described by the following equation (1).
- ka the adsorption rate coefficient
- kd the desorption rate coefficient
- c the concentration of the molecule to be analyzed near the metal surface.
- c is the ideal condition for constantly replacing the metal surface with fresh liquid.
- Is constant, and ka and kd can be obtained from the measurement results by solving a simple differential equation.
- X is the distance of the metal surface force
- D is the diffusion coefficient of the molecule to be analyzed
- c is the concentration of the molecule to be analyzed.
- the surface plasmon signal R (the difference from the surface plasmon signal when the molecule to be analyzed is adsorbed) is proportional to the surface adsorption amount of the molecule to be analyzed. It is expressed by 3).
- R represents the surface plasmon signal
- Rmax represents the signal when the analyte molecule is saturated and adsorbed.
- Equation (1), (2), and (3) can replace the difference equation approximately with the following difference equation.
- R [t] (kaXC [0, (t At)] X (Rmax— R [t— At]) — kdXR [t— At]) X At +
- C [0, t] DX (C [Ax, (t At)] — C [0, (t At)]) + C [0, (t—At)] — (kaX
- R [t] represents a surface plasmon signal at time t
- At represents a minute time.
- t represents the concentration of the molecule to be analyzed at a point X away from the metal surface force distance X at time t, and ⁇ represents a minute distance.
- Sm represents the saturated adsorption amount of the analyzed molecule.
- equations (5), (6), and (7) approximate equations (1), (2), and (3).
- the diffusion of the molecule to be analyzed at the At time must be sufficiently shorter than the distance ⁇ .
- the temporary diffusion coefficient D expressed by the following formula (8) needs to be 0.01 or more and 0.5 or less
- It is preferably 0.05 or more and 0.45 or less.
- the amount of change in At during the concentration of the analyzed molecule at the point in contact with the metal surface needs to be sufficiently small, and is expressed by the following formula (9).
- Expression (10) is described as the following Expression (11) using the influence constant Ax, Ax must be 0.1 or less and 0.0001 or more, preferably 0.03 or less and 0.001 or more. It is.
- Ax AxXD / (D XSmXka) (11)
- the surface plasmon signal R [t] can be described by using the boundary condition of the following equation (12) for the above equations (4) to (11).
- SF condition does not include the analyte molecule in the SF condition.
- R [t] (kaXC X (Rmax— R [t— At]) kdXR [t— At]) X At + R [t— At] o
- C [0, t] DX (C [Ax, (t At)] — C [0, (t At)]) + C [0, (t— At)] — (kaX C [0, (t 1 At)] X (Rmax—R [t—At]) 1 (kdXR [t— ⁇ t]) / Rmax X Sm X At)
- the diffusion coefficient D is an unknown in addition to ka, kd and Rmax.
- the diffusion coefficient D can be calculated from the molecular weight by using the Einstein equation, but in practice it is preferable to actually measure the molecular shape and the solvent greatly. Also, in the 0 ⁇ t ⁇ t region, the noise of the measurement system is large and the error increases when used in the calculation. Therefore, non-linear regression is performed on the t ⁇ t region with ka, kd, Rmax, and D as unknowns, and the curves for the 0 ⁇ t region obtained from ka, kd, Rmax, and D are compared with the measured values. As shown in Fig. 4, it shows a good agreement.
- the actual calculation may be any algorithm as long as the difference equations (13) to (17) are used. Any known method may be used for the non-linear regression method.
- the noise width of the signal change in the reference cell and the baseline fluctuation within the measurement time are measured. This makes it possible to acquire highly reliable binding detection data.
- the time for stopping the flow of the liquid is not particularly limited, but is, for example, 1 second to 30 minutes, preferably 10 seconds to 20 minutes, and more preferably about 1 minute to 20 minutes.
- the liquid in the flow path system is changed from a control liquid not containing the test substance to be measured to a sample liquid containing the test substance to be measured, and thereafter
- the change in surface plasmon resonance can be measured with the flow of the sample liquid stopped.
- a substance that interacts with a test substance is bound! /
- a reference cell and a detection cell combined with a substance that interacts with the test substance are connected in series.
- a change in surface plasmon resonance can be measured by installing in a channel system and flowing a liquid through the reference cell and the detection cell.
- the measurement is performed once for the volume (Vs ml) of the cell used for the measurement (the total volume of the cells when the reference cell and the detection cell described above are used).
- the ratio (Ve / Vs) of the liquid exchange amount (Ve ml) per unit is preferably 1 or more and 100 or less.
- Ve / Vs is more preferably 1 or more and 50 or less, and particularly preferably 1 or more and 20 or less.
- Body product of cell used in measurement (Vs ml) is not particularly limited, preferably 1 X 10- 6 ⁇ 1. Oml, particularly preferably 1 X 1 0 one 5 ⁇ 1 X 10- degree.
- the time required for exchanging the liquid is preferably from 0.01 seconds to 100 seconds, more preferably from 0.1 seconds to 10 seconds.
- the phenomenon of surface plasmon resonance is the boundary force between an optically transparent substance such as glass and the metal thin film layer.
- the intensity of the reflected monochromatic light depends on the refractive index of the sample on the metal exit side.
- the sample can be analyzed by measuring the intensity of the reflected monochromatic light.
- the surface plasmon resonance measuring apparatus used in the present invention will be described.
- a surface plasmon resonance measuring apparatus is an apparatus for analyzing the characteristics of a substance to be measured using a phenomenon in which surface plasmons are excited by light waves.
- the surface plasmon resonance measuring apparatus used in the present invention includes a dielectric block, a metal film formed on one surface of the dielectric block, a light source for generating a light beam, and the light beam to the dielectric block.
- an optical system that includes various incident angle components and an optical beam totally reflected at the interface so that a total reflection condition can be obtained at the interface between the dielectric block and the metal film.
- Photodetection means for measuring the intensity and detecting the surface plasmon resonance state.
- the dielectric block is formed as one block including all of the light beam incident surface, the light exit surface, and the one surface on which the metal film is formed, and the dielectric block includes the dielectric block.
- the metal film is integrated.
- the surface plasmon resonance measuring apparatus described in 15 can be preferably used.
- the contents described in JP-A-2001-330560 and JP-A-2002-296177 are all cited in the present specification as part of the disclosure of the present specification.
- a dielectric block, a thin film layer formed of a metal film formed on one surface of the dielectric block, and the thin film layer A plurality of measurement units each including a sample holding mechanism for holding a sample on the surface, a support that supports the plurality of measurement units, a light source that generates a light beam, and the light beam to the dielectric block.
- an optical system that is incident at various incident angles so that a total reflection condition is obtained at the interface between the dielectric block and the metal film, and the intensity of the light beam that is totally reflected at the interface are measured.
- the light reflection means for detecting the state of total reflection attenuation due to surface plasmon resonance, and the total reflection condition and various incident angles can be obtained sequentially for each dielectric block of the plurality of measurement units.
- a surface plasmon resonance measuring apparatus for measuring the state of total reflection attenuation due to surface plasmon resonance, and the total reflection condition and various incident angles.
- the optical system and the light detection means are kept stationary, and the drive means moves the support.
- the support is a turntable that supports the plurality of measurement units on a circumference around a rotation axis, and the drive means rotates the turntable intermittently. It is desirable that Further, in this case, the support is a support that supports the plurality of measurement units arranged in a straight line, and the support is intermittently provided in the direction in which the plurality of measurement units are arranged. A straight line moving object may be applied.
- the support may be kept stationary, and the driving means may move the optical system and the light detection means.
- the support body supports the plurality of measurement units on a circumference
- the driving means includes a plurality of optical systems and light detection means supported by the support body. It is desirable to rotate intermittently along the measurement unit.
- the support member is a device that supports the plurality of measurement units arranged in a straight line
- the drive unit includes the optical system and the light detection unit that are supported by the support member. It is also possible to apply one that intermittently moves linearly along the measurement unit
- the driving means rotates the rotating shaft in one direction to the plurality of measurement units.
- the pivot shaft is returned to the other direction by the same amount as the pivot amount, and then the pivot shaft is pivoted in the one direction for the next series of measurements. It is desirable that
- the plurality of measurement units are connected in a row by a connecting member to form a unit connection body, and the support body is configured to support the unit connection body. , Hope to be.
- the above-described measuring apparatus is provided with means for automatically supplying a predetermined sample to each sample holding mechanism of the plurality of measurement tubes supported by the support. It is hoped that
- the dielectric block of the measurement unit is fixed to the support, and the thin film layer of the measurement unit and the sample holding mechanism are integrated to form a measurement chip. It is desirable that the dielectric block is formed to be exchangeable.
- a force set containing a plurality of measurement chips and a measurement chip taken out from the cassette one by one are supplied in a state of being combined with the dielectric block. It is desirable to provide a chip supply means.
- the dielectric block, the thin film layer, and the sample holding mechanism of the measurement unit may be integrated to form a measurement chip, and the measurement chip may be formed to be replaceable with respect to the support.
- the measurement chip has such a configuration, a cassette that stores a plurality of measurement chips, and a chip supply that takes out the measurement chips from the cassette one by one and supplies them in a state of being supported by a support. It would be desirable to be provided with means.
- the optical system is configured to cause the light beam to be incident on the dielectric block in a state of convergent light or divergent light, and the light detection means is present in the totally reflected light beam. Desirably configured to detect dark line location due to total reflection attenuation
- the optical system is configured so that a light beam is incident on the interface in a defocused state.
- the beam diameter in the moving direction of the support at the interface of the light beam is 10 times or more the mechanical positioning accuracy of the support.
- the measurement unit is supported above the support, and the light source is disposed so as to emit the light beam downward from a position above the support, It is desirable that the optical system includes a reflecting member that reflects the light beam emitted downward toward the upper side and travels toward the interface.
- the measurement unit is supported on the upper side of the support, and the optical system causes the light beam to enter the interface from the lower side of the interface.
- the light detection means is disposed at a position above the support with the light detection surface facing downward, and reflects the light beam totally reflected at the interface upward, so that the light It is desirable to provide a reflecting member that advances toward the detecting means.
- a temperature adjusting means for maintaining the measuring unit before being supported by the support and Z or after being supported at a predetermined set temperature is provided. I hope that.
- a means for stirring the sample stored in the sample holding mechanism of the measurement unit supported by the support before detecting the state of total reflection attenuation is provided. It is desirable.
- a reference liquid supply that supplies a reference liquid having optical characteristics related to the optical characteristics of the sample to at least one of the plurality of measurement units supported by the support.
- a correction means for correcting the data indicating the total reflection attenuation state relating to the sample obtained by the light detection means based on the data indicating the total reflection attenuation state relating to the reference liquid. It is desirable to be provided Yes.
- the reference liquid supply means supplies the solvent as a reference liquid.
- the measurement apparatus described above is supplied to each of the measurement units, the mark indicating the individual identification information, the reading means for reading the mark from the measurement unit used for measurement, and the measurement unit.
- Input means for inputting sample information on the sample to be measured
- display means for displaying the measurement results, and connected to the display means, the input means and the reading means, and the individual identification information and the sample for each measurement unit
- the display means stores the information associated with the information, and associates the measurement result obtained for the sample held in a certain measurement unit with the individual identification information and the sample information stored for the measurement unit. It is desirable to provide a control means for displaying on the screen.
- the support and the optical element are detected.
- the system and the light detection means are moved relative to each other to detect the state of total reflection attenuation with respect to the sample in another measurement unit, and then the support, the optical system and the light detection means are moved relative to each other, and Measurements can be performed on samples in two measurement units by detecting the total reflection attenuation state again.
- a measurement chip used in the present invention is a measurement chip for use in a surface plasmon resonance measurement apparatus having the configuration described in the present specification, and is formed on a dielectric block and one surface of the dielectric block.
- the dielectric block is formed as one block including all of the light beam entrance surface, the light exit surface, and the one surface on which the metal film is formed, and the dielectric block includes the dielectric block.
- Metal film is integrated! /
- the metal constituting the metal film is not particularly limited as long as it can cause surface plasmon resonance.
- Preferred examples include free electron metals such as gold, silver, copper, aluminum, and platinum, with gold being particularly preferred. These metals can be used alone or in combination.
- an intervening layer having chromium equal force may be provided between the substrate and the layer having metal force.
- the thickness of the metal film is arbitrary, but for example, a surface plasmon resonance measuring apparatus was considered. In this case, it is preferably 0.1 nm or more and 500 nm or less, particularly preferably 1 nm or more and 200 nm or less. If it exceeds 500 nm, the surface plasmon phenomenon of the medium cannot be sufficiently detected. In the case where an intervening layer having chromium isotropic force is provided, the thickness of the intervening layer is preferably 0.1 nm or more and 10 nm or less.
- the metal film may be formed by a conventional method, for example, sputtering, vapor deposition, ion plating, electroplating, electroless plating, or the like.
- the metal film is preferably disposed on the substrate.
- “disposed on the substrate” means not only when the metal film is disposed so as to be in direct contact with the substrate, but also through another layer where the metal film is not directly in contact with the substrate. It is meant to include the case where it is.
- a substrate that can be used in the present invention for example, when considering use for a surface plasmon resonance measuring apparatus, generally, an optical glass such as BK7, or a synthetic resin, specifically, polymethylol methacrylate, polyethylene
- a material having a material strength that is transparent to laser light, such as terephthalate, polycarbonate, and cycloolefin polymer can be used.
- Such a substrate is preferably a material that does not exhibit anisotropy with respect to polarized light and has excellent workability.
- the metal film preferably has a functional group capable of immobilizing a physiologically active substance on the outermost surface.
- the “outermost surface” here means “the farthest side from the metal film”.
- Preferred functional groups include —OH, —SH, —COOH, —NR 2 (wherein R 1 and R 2 each independently represent a hydrogen atom or a lower alkyl group), CHO, and one NR 3 NRiR 2 (Wherein R 2 and R 3 each independently represent a hydrogen atom or a lower alkyl group), —NCO, —NCS, epoxy group, or bur group.
- the number of carbon atoms in the lower alkyl group is not particularly limited, but is generally about C1 to C10, preferably C1 to C6.
- a method for introducing these functional groups into the outermost surface for example, a polymer containing a precursor of these functional groups is coated on a metal surface or metal film, and then the outermost surface is subjected to chemical treatment. Precursor force located in the method of generating those functional groups.
- a physiologically active substance is introduced via the functional group.
- the bioactive substance can be immobilized on the metal film by covalently bonding the quality
- the physiologically active substance immobilized on the surface of the measurement chip of the present invention is not particularly limited as long as it interacts with the measurement target.
- immune proteins, enzymes, microorganisms, nucleic acids, low molecules examples include organic compounds, non-immune proteins, immunoglobulin-binding proteins, sugar-binding proteins, sugar chains that recognize sugars, fatty acids or fatty acid esters, or polypeptides or oligopeptides having ligand-binding ability.
- Examples of immunity proteins include antibodies and haptens that use the measurement target as an antigen.
- various immunoglobulins that is, IgG, IgM, IgA, IgE, and IgD can be used.
- the measurement target is human serum albumin
- an anti-human serum albumin antibody can be used as the antibody.
- pesticides insecticides, methicillin-resistant Staphylococcus aureus, antibiotics, narcotics, cocaine, heroin, cracks, etc.
- antigens for example, anti-atrazine antibodies, anti-kanamycin antibodies, anti-methamphetamine antibodies, or pathogenicity
- E. coli antibodies against O antigen 26, 86, 55, 111, 157, etc. can be used.
- various enzymes such as oxidoreductases, hydrolases, isomers, and the like are not particularly limited as long as they exhibit activity against the substance to be measured or the substance to be measured.
- a synthase, a desorbing enzyme, a synthase, etc. can be used.
- glucose oxidase can be used when the measurement object is glucose
- cholesterol oxidase can be used when the measurement object is cholesterol.
- pesticides, insecticides, methicillin-resistant Staphylococcus aureus, antibiotics, narcotics, cocaine, heroin, cracks, etc. are used as measurement objects, they exhibit a specific reaction with substances metabolized from them, such as acetylcholinesterase.
- Enzymes such as catecholamine esterase, noradrenaline esterase and dopamine esterase can be used.
- microorganism various microorganisms including Escherichia coli can be used without particular limitation.
- nucleic acid those that hybridize complementarily with the nucleic acid to be measured can be used.
- nucleic acid either DNA (including cDNA) or RNA can be used.
- DNA including cDNA
- RNA can be used.
- DNA The type is not particularly limited and may be any of naturally-derived DNA, recombinant DNA prepared by gene recombination technology, or chemically synthesized DNA.
- Examples of the low molecular weight organic compound include any compound that can be synthesized by an ordinary organic chemical synthesis method.
- the non-immune protein is not particularly limited, and for example, avidin (streptavidin), piotin or a receptor can be used.
- immunoglobulin-binding protein for example, protein A or protein G, rheumatoid factor (RF) and the like can be used.
- RF rheumatoid factor
- sugar-binding proteins examples include lectins.
- fatty acid or fatty acid ester examples include stearic acid, arachidic acid, behenic acid, ethyl stearate, ethyl arachidate, and ethyl behenate.
- the physiologically active substance is a protein or nucleic acid such as an antibody or an enzyme
- the immobilization is performed by covalently bonding to a functional group on the metal surface using an amino group, a thiol group, or the like of the physiologically active substance. It can be carried out.
- the measurement chip on which a physiologically active substance is immobilized as described above can be used for detection and Z or measurement of a substance that interacts with the physiologically active substance.
- At least a measurement chip (cell) in which a physiologically active substance is covalently bonded to the surface is used, and the sample liquid containing the test substance to be measured is brought into contact with the cell.
- test substance for example, a sample containing a substance that interacts with the physiologically active substance described above can be used.
- the surface plasmon resonance measuring apparatus shown in Fig. 1 slidably engages with two guide rods 400, 400 arranged in parallel to each other as a support for supporting the measurement unit, and along the drawing,
- the slide block 401 which can be linearly moved in the Y direction, is used.
- the slide block 401 is screwed with a precision screw 402 arranged in parallel with the guide rods 400, 400 so that the precision screw 402 is rotated forward and backward by the pulse motor 403 constituting the support driving means together with the precision screw 402. Become! /
- the driving of the pulse motor 403 is controlled by a motor controller 404. That is, an output signal S40 of a linear encoder (not shown) that is incorporated in the slide block 401 and detects the position of the slide block 401 in the longitudinal direction of the guide rod 400, 400 is input to the motor controller 404.
- the controller 404 controls the driving of the pulse motor 403 based on the signal S40!
- a laser light source 31 a condensing lens 32, and a photodetector 40 are arranged so that a slide block 401 that moves along the guide rods 400 is sandwiched between the left and right sides. ing.
- the condenser lens 32 condenses the light beam 30.
- a photodetector 40 is installed.
- a stick-like unit coupling body 410 formed by connecting and fixing eight measurement units 10 is used, and the eight measurement units 10 are slid in a line. It is set in block 401.
- FIG. 2 shows the structure of the unit connector 410 in detail.
- the unit connection body 410 is formed by connecting eight measurement units 10 by connection members 411.
- This measurement unit 10 is formed by integrally forming the dielectric block 11 and the sample holding frame 13 from, for example, transparent resin, and constitutes a measurement chip that can be exchanged for the turntable. ing. In order to make the exchange possible, for example, the measurement unit 10 may be fitted and held in a through hole formed in the turntable. In this example, the sensing substance 14 is fixed on the metal film 12. [0100] Example: Measurement with liquid stopped
- the dielectric block of the present invention in which 50 nm of gold is deposited as a metal film, is treated with Mode ⁇ ⁇ 208UV-zone cleaning system (TECHNOVISION INC.) For 30 minutes, and then 11 hydroxy-1 in ethanol Z water (80/20). A 5.0 mM solution of ndecanethiol was added so as to contact the metal film, and surface treatment was performed at 25 ° C for 18 hours. Thereafter, washing was performed 5 times with ethanol, once with an ethanol / water mixed solvent, and 5 times with water.
- Mode ⁇ 208UV-zone cleaning system TECHNOVISION INC.
- a mixed solution 701 of 200 mM EDC and 50 mM NHS was added and left for 10 minutes. After removing the mixed solution, it was washed 3 times with 10 liters of water and 3 times with liters of Acetate 5.0 buffer. The chip was replaced with 1M ethanolamine solution and left for 10 minutes. The inside of the chip was washed 10 times with 100 ⁇ l of Acetate 5.0 buffer.
- a cell having an internal volume of 151 was created by covering the ProteinA-fixed chip of the present invention with a dielectric block covered with silicon rubber. Two channels of 1mm diameter were drilled in the silicon rubber of the lid, and a flow path was created through a Teflon tube with an inner diameter of 0.5mm and an outer diameter of lmm. Similarly, a flow path was created by connecting the reference chip with a flow path and connecting the two chips in series. Two chips of this flow path system are installed in the surface plasmon resonance measuring apparatus of the present invention.
- HBS-EP buffer manufactured by BIAcore.
- the composition of HBS-EP buffer is HEPES (N-2-Hydroxyethylpiperazine-N'-2-ethanesulfonic Acid) 0.01mol / l (pH7.4), NaC10.15mol / l, EDTA 0.003mol / l, Surfactant P20 0.005weight 0/0 Ru der.
- HEPES N-2-Hydroxyethylpiperazine-N'-2-ethanesulfonic Acid
- mouse IgG solution mouse IgG (purchased from Cosmo Bio) dissolved in HBS-EP buffer so as to be 10 ⁇ g / ml) at a rate of 20 / zl / sec.
- the time required for the replacement was 5 seconds.
- the following experiment was performed using BIAcore3000 (manufactured by BIAcore).
- the sensor chip CM-5 was set by a normal method and then primed with HBS-EP buffer.
- a buffer and various solutions were flowed through the flow cell 2 at a flow rate of 10 1 / min.
- a mixed solution of 200 mM EDC and 50 mM NHS was allowed to flow for 7 minutes, and then washed with HBS-EP buffer.
- Protein A solution Protein A (Protein A (manufactured by Nacalai Tester) dissolved in Acetate 5.0 (manufactured by BIAcore) so as to be 10 ⁇ g / ml) was flowed for 1 minute to immobilize Protein A.
- a 1M ethanolamine solution was allowed to flow for 7 minutes.
- the ethanolamine solution was washed with HBS-EP buffer.
- the amount of resonance signal change due to protein A fixation was 300 RU.
- mice IgG solution mouse IgG (purchased from Cosmo Bio) dissolved in HBS-EP buffer at 10 g / ml) was injected with the kinject command, and signals were measured for 5 minutes each for binding and dissociation.
- the measured signal was subjected to fitting in 1: 1 (Langmuir) binding mode using analysis software BIAevaluation 3.1 (manufactured by BIAcore) to obtain ka, kd, and Rmax.
- Table 1 shows the values of the parameters obtained in the examples and comparative examples. It can be seen that the values of ka and kd obtained in the example are close to the values obtained in the comparative example. In addition, the dissociation constant in a state where the baseline fluctuation was good could be obtained in the same way as in the ideal state.
- the present invention it is possible to obtain a highly reliable measurement result in which the noise level (noise width of the reference chip) is low and the baseline change (signal change of the reference chip) is small.
- the method of measuring the signal change of surface plasmon resonance with the liquid flow stopped is fixed to the metal surface in surface plasmon resonance. It is now possible to calculate the dissociation constants of molecules that interact with the analyzed molecules.
- FIG. 1 shows a surface plasmon resonance measuring apparatus used in Examples.
- 10 is a measurement unit
- 30 is a light beam
- 31 is a laser light source
- 32 is a condenser lens
- 40 is a photodetector
- S40 is an output signal
- 400 is a guide rod
- 401 is a slide block
- 402 is precision Screw
- 403 is a pulse motor
- 404 is a motor controller
- 410 is a unit connection.
- FIG. 2 shows a dielectric block used in the example.
- 10 is a measurement unit
- 11 is a dielectric block
- 12 is a metal film
- 13 is a sample holding frame
- 14 is a sensing substance
- 410 is a mute connector
- 411 is a connection member.
- Figure 3 shows a comparison of ideal conditions, stop flow conditions, and measured values for SPR measurements.
- FIG. 4 shows the result of comparing the curve of the entire region obtained by the method of the present invention, ka, kd, Rmax, and D force, and the measured value of SPR.
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Abstract
Description
Claims
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EP05780889A EP1795886A4 (en) | 2004-08-24 | 2005-08-24 | METHOD FOR CALCULATING THE DISSOCIATION CONSTANT IN SURFACE PLASMON RESONANCE ANALYSIS |
JP2006531936A JP4664298B2 (ja) | 2004-08-24 | 2005-08-24 | 表面プラズモン共鳴分析における解離定数の算出方法 |
US11/660,958 US7602495B2 (en) | 2004-08-24 | 2005-08-24 | Method for measuring dissociation constant by surface plasmon resonance analysis |
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JP2004244151 | 2004-08-24 | ||
JP2004-244151 | 2004-08-24 |
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PCT/JP2005/015318 WO2006022277A1 (ja) | 2004-08-24 | 2005-08-24 | 表面プラズモン共鳴分析における解離定数の算出方法 |
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US (1) | US7602495B2 (ja) |
EP (1) | EP1795886A4 (ja) |
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WO (1) | WO2006022277A1 (ja) |
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US7449343B2 (en) * | 2004-09-07 | 2008-11-11 | Fujifilm Corporation | Method for measuring surface plasmon resonance |
US20070176728A1 (en) * | 2006-01-31 | 2007-08-02 | Ranganath Tirumala R | Tiled periodic metal film sensors |
EP3102708B1 (en) | 2014-02-05 | 2020-11-18 | Deakin University | Aptamer construct |
TW202214869A (zh) | 2020-08-18 | 2022-04-16 | 西班牙商雷勁那賽克斯公司 | 用於檢測 sars-cov-2 刺突蛋白之組合物及方法 |
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JP4664298B2 (ja) | 2011-04-06 |
EP1795886A1 (en) | 2007-06-13 |
EP1795886A4 (en) | 2010-04-07 |
US7602495B2 (en) | 2009-10-13 |
US20080037023A1 (en) | 2008-02-14 |
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