JP2006337244A - Method and apparatus for measuring sample by surface plasmon resonance method - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method and an apparatus for analyzing samples including at least two samples or more on the same sensor chip in a surface plasmon resonance sensor device that utilizes external oscillations. <P>SOLUTION: By constituting spacer molecules which fix ligands to be different, frequency characteristics are changed for each spacer molecule. It is possible to measure a plurality of types of a large number of samples including analyte molecules which bond, according to each ligand molecule on the same sensor chip by the differences in the frequency characteristics. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to an apparatus capable of measuring a sample including at least two samples of interaction between a ligand molecule and an analyte molecule by a surface plasmon resonance apparatus that applies vibration from outside.

The surface plasmon resonance (SPR) method is performed when the refractive index of a medium such as a solution in contact with the lower surface of a sensor chip (a metal substrate such as gold deposited on a glass substrate) changes. This is a method of measuring the change in the refractive index of the medium by utilizing the fact that the angle of the reflected light with reduced intensity among the reflected light incident at various angles through the prism is changed. For example, when a ligand is bonded to the surface of the metal thin film and the ligand is bonded to the analyte, the dielectric constant near the metal thin film changes. A method for detecting the change by utilizing the surface plasmon resonance phenomenon and analyzing the binding amount of the ligand and the analyte is known. (For example, refer nonpatent literature 1). In addition, a technique (for example, refer to Patent Document 1) that controls the separation and movement of a sample on a metal thin film by applying an electric field to the back surface of the metal thin film on which a ligand is immobilized, or a change in the refractive index of the metal thin film is used. Thus, a technique for measuring a large number of samples at once has been proposed (see, for example, Patent Document 2). However, in these methods, the amount of analyte bound to the ligand is analyzed from a minute amount of change in angle at which the amount of light decreases as surface plasmons are excited. In addition, a method for analyzing an analyte bound to a ligand from a frequency characteristic of a surface plasmon resonance angle with respect to an application means for applying external vibration to a region where the ligand is immobilized has been proposed.
Anal. Chem. 1998, 70, 2019-2024. JP 2003-65947 A JP 2002-75336 A

  Conventionally, when performing multi-item measurement using a surface plasmon resonance sensor, it has been necessary to immobilize a sensing molecule corresponding to the measurement item, for example, an antibody, to a sensor that is independent for each item. A two-dimensional SPR sensor has been proposed to perform simultaneous multi-item measurement, but there were problems in terms of antibody immobilization and measurement sensitivity. An object of the present invention is to provide a surface plasmon resonance sensor by external vibration capable of simultaneous multi-item measurement on the same sensor.

  In order to solve the above problems, an immunoassay device using external vibration according to the present invention includes means for irradiating measurement light, means for receiving reflected light or transmitted light of the measurement light, and external vibration in a fixed ligand region. And means for measuring the frequency characteristics of the direction change of the ligand molecules, and measuring the frequency characteristics of the angular fluctuations that cause surface plasmon resonance while applying external vibration. The frequency characteristics are determined by the molecular weight of the analyte, the amount of binding, and the type of spacer immobilizing the ligand. That is, in the measurement of the low molecular weight and the high molecular weight, since the molecular weight of the analyte is different and the frequency characteristics are different, simultaneous measurement is possible. When there is little difference in the molecular weight of the analyte, it is possible to change the frequency characteristics by using the mass difference of the ligand spacer, and to provide a surface plasmon resonance sensor by external vibration capable of simultaneous multi-item measurement.

  In the analysis method and apparatus of the present invention, external vibration is applied, and the frequency characteristic of the surface plasmon resonance angle is obtained by the difference in the molecular weight of the analyte with respect to the external vibration. As described above, the frequency characteristic varies depending on the difference in molecular weight, so that the analyte can be measured on the same sensor. In addition, for the measurement of an analyte having the same molecular weight, multiple samples can be measured by changing the mass of the spacer.

  Hereinafter, embodiments of the immunoassay apparatus of the present invention will be described in detail with reference to the drawings.

  FIG. 1 shows an optimal form of an immunoassay apparatus according to the first embodiment of the present invention. In FIG. 1, 1 is a light source, 2 is a prism, 3 is incident light, 4 is a light receiving device, 5 is reflected light, 6 is an SPR resonance angle, 7 is a metal thin film and upper electrode, 8 is a lower electrode, and 9 is a metal thin film. Ligand molecules fixed to the upper electrode, 10 is an AC source, 11 and 12 are active filters that pass only a specific frequency, 13 is the phase of external vibration (θ0), and the phase of the signal component of external vibration included in the reflected light A detector for comparing (θ1), 14 is an A / D converter that converts an analog signal obtained by the detector 13 into a digital signal, and 15 is an external vibration. Reference numeral 16 denotes an analyte in the sample. The light source 1 is disposed at a position where the prism 2 is irradiated with light under the condition of total reflection. A ligand 9 is fixed to the back surface of the irradiated surface of the metal thin film / upper electrode 7 to be irradiated. The light receiving device 4 is disposed at a position where the reflected light 5 reflected by the prism 2 can be received. The lower electrode 8 is arranged so that external vibration can be applied to the ligand 9 fixed to the metal thin film / upper electrode 7. In FIG. 1, the metal thin film / upper electrode 7 is disposed on the upper side of the apparatus, and the lower electrode 8 is disposed on the lower side of the apparatus. Needless to say. The intensity of the reflected light 5 including the SPR resonance angle 6 is detected using the light receiving device 4. Further, an AC source 10 is connected to the metal thin film / upper electrode 7 and the lower electrode 8. An external vibration 15 having a frequency f is applied to the metal thin film / upper electrode 7 by the AC source 10. The ligand 9 vibrates due to external vibration. By this vibration, the dielectric constant of the ligand 9 is changed, and the change in the resonance angle is detected by the light receiving device 4. The active filter 12 removes components other than the external vibration signal component, and the detector 13 compares the phase of the external vibration and the phase of the signal component of the external vibration included in the reflected light. As the light source 1, a He—Ne laser, an Ar laser, a dye laser, or the like can be used. For example, an AlGaAs double heterojunction visible light semiconductor laser (for example, manufactured by ROHM), a collimator lens (for example, Matsushita) Nitto Co., Ltd.) and a polarizing beam splitter (for example, Sigma Koki Co., Ltd.). As the prism, a conical shape, a hemispherical shape, or the like can be used. The light receiving device 4 is a device that converts the intensity of the reflected light 5 into a voltage by using an operational amplifier (for example, manufactured by National Semiconductor) and a resistance element for a CCD, Si, PIN, photodiode (for example, manufactured by Hamamatsu Photonics) or the like. It may be.

  FIG. 2 shows the quantitative method. 2A is obtained by comparing the phase of the signal component of the receptor vibration 12 included in the reflected light with the external vibration device 8 shown in FIG. By detecting the inflection point (following frequency limit) of the frequency characteristic, it becomes possible to obtain a result with high measurement accuracy. FIG. 2B shows the result of measuring the inflection point of the frequency characteristic as a time change. By measuring the time change of the inflection point, the amount of the analyte bound to the ligand can be measured. As the combined analyte increases, the time change of the inflection point becomes longer from t1 to t3. Quantification is possible because the time change and the combined analyte are proportional.

FIG. 3 shows a method for creating a sensor film. Alkylthiol is a molecule that constitutes a self-assembled film (SAM) on a metal film. In this implementation, the ligand was immobilized using 11-Mercaptoundecanoic acid (manufactured by Aldrich) as a spacer. The preparation method is as follows. A commercially available SPR sensor chip (a glass plate having a gold film thickness of 50 nm and a size of 9 mm × 9 mm) prepared by preparing a 2.5 mM ethanol solution of thiol molecules and forming a gold film on a glass substrate in each solution. SAM was formed by immersion for 24 hours at room temperature. After forming the SAM film, it was washed with an ethanol solution to obtain an SPR sensor chip. FIG. 3B shows sensor films having different thiol molecule configurations. In addition, SH- (CH2) n-R can be used to create FIG. n is an integer of 1 to 30, and R is a carboxyl group or an amino group. FIG. 3C shows the basic structure of a thiol molecule that can be used as a SAM. Next, a method for immobilizing a ligand molecule is shown. The antibody was bound to the carboxyl group of the SAM thiol molecule of the sensor chip prepared above. This operation was specifically performed as follows. 0.4M EDC (manufactured by Aldrich) /0.1M NHS (manufactured by Aldrich), (EDC: N-ethyl-N ′-(3-dimethylaminopropyl) carbodiimide hydrochloride, NHS: N-hydroxysuccinimide) The carboxyl group was activated. EDC / NHS was dissolved in ultrapure water (Mili Q water). The activation time was 7 minutes. The activated carboxyl group was coupled using the amino group of the protein. The concentration of bovine serum albumin antibody (Funakoshi) was 100 μg / ml and dissolved in 10 mM acetate buffer pH 4.5 (Wako Pure Chemical Industries). The immobilization time was 7 minutes. Finally, the unreacted activated carboxyl group was inactivated using 1M ethanolamine pH 9.0 (manufactured by Wako Pure Chemical Industries, Ltd.).

  FIG. 4 shows a conceptual diagram of simultaneous determination of a low molecule (drug etc.) and a high molecule (protein etc.). The sensor unit is a sensor chip composed of only a single spacer in FIG. The correlation between the external vibration (f) and the measurement time (t) will be described below with reference to FIG. Aspirin (molecular weight 180) (manufactured by Aldrich) is used as the low molecule, and bovine serum albumin (66 kDa) (manufactured by Aldrich) is used as the analyte. An anti-aspirin antibody (manufactured by Funakoshi) and an anti-bovine serum albumin antibody (manufactured by Funakoshi) are immobilized on the sensor chip of FIG. The ligand molecules are immobilized on the same sensor using the same spacer, and the interaction of substances having different molecular weights of the analyte is shown in FIG. 4B, and the frequency characteristic at this time is shown in FIG. 4A. The frequency characteristic of bovine serum albumin is obtained as a follow-up frequency limit f2 shown in FIG. This is because the mass of the ligand molecule increases due to binding of a polymer substance such as bovine serum albumin to the ligand molecule, making it impossible to follow external vibration. On the other hand, in a low molecular weight compound such as aspirin, since the mass increase of the ligand molecule is small, it is possible to follow up to a higher frequency than that of bovine serum albumin. Therefore, the frequency characteristics are shown in FIG. Obtained as the frequency limit f1. Therefore, when the difference in the molecular weight of the analyte is large, it is possible to obtain the tracking frequency limit of the low molecule and the high molecule on the same sensor.

FIG. 5 shows a conceptual diagram when the tracking frequency is measured by sensor chips having different spacer lengths. FIG. 5A is a tracking frequency characteristic diagram obtained at the time of simultaneous determination of bovine serum albumin (66 kDa) and human serum albumin (68 kDa). The follow-up frequency limit f1 is the frequency of bovine serum albumin, and the follow-up frequency limit f2 is human serum albumin. As shown in FIG. 5B, since the spacers have different lengths, different frequency characteristics can be obtained. The follow-up frequency limit f1 shown in FIG. 5 (b) immobilized bovine serum albumin antibody, and the follow-up frequency limit f2 immobilized human serum albumin antibody. When the pendulum principle is used, the length of the spacer becomes n ^1/2 times as long as the spacer becomes 1 / n, and the shorter the spacer, the higher the frequency. Since various frequency characteristics can be obtained, simultaneous quantification is possible.

  FIG. 6 shows a conceptual diagram when the following frequency is measured when the molecular weight of the analyte is almost the same and the spacer mass is different. FIG. 4 is a conceptual diagram obtained when ligand molecules are immobilized on the sensor created in FIG. 3B and external vibration is applied. When ovalbumin (45 kDa), bovine serum albumin (66 kDa), and human serum albumin (68 kDa) are measured as shown in FIG. 6 (b), the frequency characteristics are obtained as shown in FIG. 6 (a). Therefore, it is difficult to measure the tracking frequency when there is no change in the spacer mass. This is because there is no difference in tracking frequency characteristics when the molecular weight of the analyte is the same and the mass of the spacer is the same. Therefore, it is possible to change the follow-up frequency of the ligand by using the sensor chip shown in FIG. 6D having different spacer densities. In L: SH- (dextran) -COOH used for SAM, when glucose is used as a constituent of L dextran, the weight ratio of S: SH- (CH2) -COOH is about 10 times, and the L chain is more Become heavier. This weight ratio becomes larger as the thiol chain becomes longer, and the weight ratio of the spacer can be arbitrarily changed by changing the polymerization ratio of dextran. If it is desired to make the chain slightly heavier than the S chain, the weight can be changed by using the M chain.

FIG. 7A shows a graph in which the vertical axis represents the amplitude (A) and the horizontal axis represents the tracking frequency limit (f). The amplitudes of f3, f2, and f1 indicate the tracking frequency limits of the spacers L, M, and S of the sensor chip in FIG. When the frequency of F3 is applied, L cannot follow, so the amplitude of L decreases, and this decrease corresponds to A1 in FIG. 7A, and the state at this time is as shown in FIG. 7B-1. Become. Subsequently, since M cannot follow when the frequency f2 is applied, the amplitude of M decreases, and this decrease corresponds to A2 in FIG. 7A, and the state at this time is shown in FIG. 7B-2. It becomes. Similarly, when the frequency of f1 is applied, S cannot follow, so the amplitude of S decreases, and this decrease corresponds to A3 in FIG. 7A, and the state at this time is shown in FIG. It becomes. Further, the force F received by the molecule at this time is represented by the following equation. F = 1 / 2ρSV ² , where ρ is the density of the spacer, S is the cross-sectional area of the spacer, and V is the velocity of the analyte. Therefore, the larger the density of the spacer, the higher the density, the analyte coupled to the L chain, which receives force F and cannot follow external vibration, is heavier and has a higher density. It becomes low and becomes the follow-up frequency limit f3 shown in FIG. Since the lightest spacer S chain has a low density, it is affected by external vibrations to the end, and the frequency characteristics are higher than those of the L and M chains. The M chain indicates an intermediate value between L and S. As the L, M, and S chains become unable to follow, the amplitude width decreases, and the respective frequency characteristics are obtained. Therefore, the density varies depending on the mass of the spacer, and a large number of tracking frequency limits can be obtained. Simultaneous multi-item measurement can be performed on the same chip.

As described above, the analysis method and apparatus of the present invention measures the frequency characteristics of the surface plasmon resonance angle with respect to external vibration. By obtaining different frequency characteristics depending on the measured substance, the analysis method and apparatus of the present invention can be used on the same sensor that has been difficult in the past. Multi-item measurement is possible. Therefore, the analysis method and apparatus of the present invention are useful for analyzing multiple items in a sample, and are useful in fields such as biology, medicine, pharmacy, and agriculture, for example.

Schematic diagram showing an example of the apparatus of the present invention Illustration of quantitative method using inflection point (following frequency limit) The figure which shows the block diagram and structural formula of the sensor chip of this invention, (a) The block diagram of the sensor chip by the same thiol molecule, (b) The block diagram of the sensor chip by the thiol molecule from which the structure of a spacer differs, (c) Sensor chip Showing the structural formula of thiol molecules that can constitute Conceptual diagram of simultaneous measurement of analytes with different molecular weights, (a) Diagram showing frequency characteristics obtained from substances with different molecular weights, (b) Conceptual diagram on sensor surface Configuration diagram of sensor chips with different spacer lengths, (a) Diagram showing frequency characteristics obtained from comparable analytes, (b) Conceptual diagram of sensor chips Analytical measurement diagram with no difference in molecular weight, (a) Diagram showing frequency characteristics obtained from the sensor chip of the same spacer, (b) Conceptual diagram on the sensor surface at that time, (c) When the type of spacer is changed The figure which shows the frequency characteristic which is obtained, (d) The conceptual diagram on the sensor surface The figure which shows the amplitude change when changing the kind of spacer, (a) The change figure of the amplitude obtained when changing the kind of spacer, (b-1) The conceptual diagram on the sensor surface in amplitude A1, (b- 2) Conceptual diagram on the sensor surface at amplitude A2, (b-3) Conceptual diagram on the sensor surface at amplitude A3

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 Light source 2 Prism 3 Incident light 4 Light-receiving device 5 Reflected light 6 SPR resonance angle 7 Metal thin film / upper electrode 8 Lower electrode 9 Ligand molecule 10 High frequency source 11, 12 Active filter 13 Phase detector 14 A / D converter 15 External vibration 16 Analyte molecule

Claims (14)

In the surface plasmon resonance method, an external vibration is applied to the sensor surface, and the interaction between the analyte molecule as the measurement molecule in the sample and the ligand molecule that binds to the analyte molecule is analyzed based on the frequency characteristics of the resonance angle with respect to the external vibration. A method for measuring a sample, characterized in that the spacer molecules for immobilizing the ligand molecules have different structures. The method according to claim 1, wherein the spacer molecules are self-assembled films having thiols or disulfides and have different chain lengths. The method of claim 1, wherein the spacer molecules have different molecular densities. In the surface plasmon resonance method, an external vibration is applied to the sensor surface, and the interaction between the analyte molecule as the measurement molecule in the sample and the ligand molecule that binds to the analyte molecule is analyzed based on the frequency characteristics of the resonance angle with respect to the external vibration. A method for measuring a sample, characterized in that the analyte molecules have different molecular weights. By measuring the amplitude reduction width, it is possible to measure a sample including at least two samples on the same sensor without identifying the analyte according to claim 2 or claim 3. The method according to claim 1 or claim 4. The method according to claim 1, wherein at least one of the analyte and the ligand is charged. The method according to claim 1 or 4, wherein the applying means is means for applying at least one of electrical and mechanical vibrations. 5. The method according to claim 1 or 4, wherein the amount of the analyte in the sample bound to the receptor is analyzed. The method according to claim 1 or 4, wherein the bound analyte substance is a protein or a peptide. The method according to claim 1 or 4, wherein the bound reactant is an antibody or an antigen-binding fragment thereof. 2. The method according to claim 1, wherein the analysis unit further includes a comparison unit that compares a phase of the external vibration with a phase of a signal component of the external vibration included in the reflected light. A method in which the step of obtaining is performed by comparing the phase of the external vibration with the phase of the signal component of the external vibration included in the reflected light and detecting the inflection point of the frequency characteristic by the comparison unit. A method for measuring a sample, comprising at least two or more samples using 8. The method according to claim 7, wherein the applying means is means for applying at least an electrical vibration, wherein the physical properties of the ligand can be further analyzed from the analyzing means. The apparatus according to claim 12, wherein the analyzing means further includes a comparing means, wherein the comparing means compares the phase of the external vibration with the phase of the signal component of the external vibration included in the reflected light, An apparatus capable of detecting an inflection point of the frequency characteristic. 14. The apparatus according to claim 13, further comprising a measuring means, wherein the measuring means can detect the degree of binding between the analyte and the ligand by measuring the time change of the inflection point of the frequency characteristic. The device characterized by being.

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