JP2008122105A - Elastic wave sensor and detection method - Google Patents

Abstract

The conventional system is suitable for experiments in a laboratory, but has a problem that it is inconvenient outside the laboratory because it requires a solution flow system and a thermostatic bath. Therefore, an acoustic wave sensor capable of solving such problems and detecting an antigen-antibody reaction by a simple method and a detection method thereof are provided.
In a detection method for detecting a substance to be measured based on a change in propagation characteristics of an elastic wave sensor, an excitation process for generating a transverse wave type surface acoustic wave and a reference solution that is a solution for diluting the substance to be measured A first phase measuring step for measuring the phase angle before dropping the measurement substance on the measurement acoustic wave element on which the substance to be measured is immobilized, and measuring the phase angle after dropping the reference solution on the measurement acoustic wave element A second phase measurement step, and an immobilization amount estimation step of estimating an immobilization amount of the substance to be measured based on the phase angle measured in the first and second phase measurement steps.
[Selection] Figure 1

Description

  The present invention relates to an elastic wave sensor used for sensing a substance to be measured and a method for detecting the substance to be measured.

  In recent years, in various fields such as surface acoustic wave medicine, environment, biotechnology, etc., a sensor using an acoustic device such as a crystal resonator, which has a simple structure and can be expected to be miniaturized, detects the amount, concentration, etc. Used to make measurements.

  An example of a sensor using an acoustic device is a solution sensor. In this solution sensor, an excitation electrode and a receiving electrode in the shape of comb-like electrode fingers are formed on a piezoelectric substrate with a sensing region interposed therebetween. When a high frequency signal is applied to the excitation electrode, an electric field is generated between the electrode fingers, and the piezoelectric substrate is excited by the piezoelectric effect to generate a surface acoustic wave.

  When a solution containing a substance to be measured is dropped into this sensing area, the propagation characteristics (for example, surface wave velocity) of surface acoustic waves propagating on the surface of the piezoelectric substrate change due to the presence of this solution. It is possible to sense a substance to be measured by detecting a change in wave propagation characteristics.

  As a solution sensor, there is an immunosensor using an antigen-antibody reaction disclosed in Patent Document 1. In this immunosensor, a sensitive film sensitive to a substance to be measured is formed between the excitation electrode and the receiving electrode. Patent Document 1 discloses a solution flow system in which a buffer solution, a latex solution modified with an antigen, and a sample solution are fed into a measurement cell equipped with an immunosensor in an immunosensor system using a Lamb wave mode acoustic device. Has been. Furthermore, in order to stabilize the reaction, a sensor, a measurement cell, and a solution flow system are installed in the thermostat.

JP-A-5-232114

  The above-described immunosensor system is suitable for experiments in a laboratory, but has a problem that it is inconvenient to use outside the laboratory because it requires a solution flow system and a thermostatic bath. Accordingly, an object of the present invention is to solve such problems and provide an elastic wave sensor capable of detecting a weight change due to an antigen-antibody reaction by a simple method and a detection method thereof.

  In order to achieve the above object, a detection method according to the present invention uses an acoustic wave element for measurement in which a measurement sensitive film exhibiting adsorptivity to a substance to be measured is formed on a piezoelectric substrate. In a detection method for detecting a substance to be measured by a change in propagation characteristics of a surface acoustic wave caused by a change in a measurement sensitive film, an excitation process for exciting a piezoelectric substrate to generate a transverse wave type surface acoustic wave, The first phase measurement step for measuring the phase angle before dropping the reference solution, which is a solution for diluting the measurement substance, onto the measurement acoustic wave element on which the substance to be measured is immobilized, and the elasticity for measuring the reference solution A second phase measurement step for measuring the phase angle after dropping on the wave element, and an immobilization amount for estimating the immobilization amount of the substance to be measured based on the phase angles measured in the first and second phase measurement steps And an immobilization amount estimation step comprising: The phase angle change due to the known immobilized amount that is because the measurement, a phase angle change obtained by the measurement, and estimates based on.

  In addition, the detection method according to the present invention uses a measurement acoustic wave element in which a measurement sensitive film exhibiting adsorptivity to a substance to be measured is formed on a piezoelectric substrate, and accompanies a change in the measurement sensitive film. In the detection method for detecting the substance to be measured by the change in the propagation characteristics of the surface acoustic wave, the reference acoustic wave element on which the same sensitive film as the measurement sensitive film is formed and the measurement acoustic wave element are the same piezoelectric. An excitation process that is provided on a conductive substrate and generates a transverse wave type surface acoustic wave by exciting a piezoelectric substrate, and a measurement elasticity in which a measurement substance is fixed to a reference solution that is a solution for diluting the measurement substance A third phase measuring step for measuring the phase angle of both acoustic wave elements when dropped on the wave element, and a phase to be measured based on the phase angle measured by the both acoustic wave elements measured in the third phase measuring step Immobilization amount estimator to estimate the amount of material immobilization When, wherein the immobilized amount estimating step is characterized the phase angle change due to the known immobilized amount that has been previously measured, and phase angle change obtained by measurement, to estimate based on.

  Furthermore, an acoustic wave sensor according to the present invention is an acoustic wave sensor having a measurement acoustic wave element on which a measurement sensitive film having adsorptivity to a substance to be measured is formed. A solution for diluting a substance to be measured, having a measurement electrode on which a measurement sensitive film is formed, and a reception electrode for receiving a transverse type surface acoustic wave propagated through the measurement region. An outer wall surrounding the measurement sensitive film on the piezoelectric substrate is provided so that the measurement sensitive film is completely covered by a certain reference solution.

  Furthermore, in the acoustic wave sensor according to the present invention, the phase angle is measured before the reference solution is dropped on the measurement acoustic wave element, and the phase angle is measured after the reference solution is dropped on the measurement acoustic wave element. And an immobilized amount estimating means for estimating an immobilized amount of the substance to be measured immobilized on the measurement sensitive film based on the phase angle measured by the phase measuring means. Is estimated based on a phase angle change due to a known fixed amount measured in advance and a phase angle change obtained by measurement.

  Furthermore, in the acoustic wave sensor according to the present invention, the reference acoustic wave element having the same sensitive film as the measurement sensitive film and the measurement acoustic wave element are provided on the same piezoelectric substrate, and the reference solution is Phase angle measuring means for measuring the phase angle of both acoustic wave elements when dropped on the acoustic wave element for measurement, and the measurement object fixed to the measurement sensitive film based on the phase angle measured by both acoustic wave elements An immobilization amount estimating means for estimating an immobilization amount of the substance, and the immobilization amount estimation means includes a phase angle change due to a known immobilization amount measured in advance, and a phase angle change obtained by measurement. , Based on the estimation.

  By using the present invention, the antibody concentration of the antibody solution immobilized on the acoustic wave sensor is proportional to the change in the phase angle of SH-SAW, so that the antigen-antibody reaction can be detected by a simple method.

  Hereinafter, embodiments of the present invention (hereinafter referred to as embodiments) will be described with reference to the drawings.

(First embodiment)
FIG. 1 shows an acoustic wave sensor 30 having a measurement acoustic wave element 30a and a reference acoustic wave element 30b, a signal generator 11, a phase angle detection circuit 12, and a reference for explaining the principle of the present embodiment. An elastic wave sensor system 10 including a solution dropping device 13 is shown.

  The acoustic wave sensor 30 is provided with a reaction field in which a sensitive film 22 is formed on a surface acoustic wave propagation path between the comb-tooth input electrode 14 and the comb-tooth output electrode 16. The structure of the reaction field uses an electrically short-circuited element of full-surface gold (Au) that is not affected by electrical properties such as conductivity of the solution. Further, in order to completely cover the sensitive film 22 with the solution, the outer wall 21 was formed by photolithography using a photosensitive epoxy resin, but the propagation loss was 3 dB or less.

  The substrate 25 of the acoustic wave sensor uses a 37 ° rotation Y-cut quartz substrate having good temperature characteristics, and the surface acoustic wave propagates in the vertical X-axis direction on the quartz substrate (Shear Horizontal-SAW: SH-SAW) is used. SH-SAW is characterized in that there is no longitudinal wave radiation to the solution dropped on the surface and there is little propagation attenuation. In this embodiment, for example, by using SH-SAW with a center frequency of 250 MHz, the wavelength λ is An elastic wave sensor that generates a transverse wave having a slight change at about 20 μm was used. In FIG. 1, the measurement acoustic wave element 30a and the reference acoustic wave element 30b have the same structure, thereby canceling changes in temperature and humidity.

  FIG. 2 shows a case where an antibody solution having a different concentration is immobilized on an elastic wave element for measurement and then a reference solution is dropped on the elastic wave element for measurement, and the phase angle of the reference elastic wave element not dropped is used as a reference. It is the characteristic view which showed the change of the phase angle with time progress of.

  As a reference solution used in the experiment, a PBS buffer solution of 10 mM, pH 7.4 was used. There are five types of solutions to be measured, ranging from 0.106 μg / ml to 1060 μg / ml by diluting a CRP antibody (Oriental Yeast Co, Ltd) with a standard solution. Next, 20 μl of each of the five types of solutions to be measured is dropped on the measurement acoustic wave element 30a and the reference acoustic wave element 30b of each acoustic wave sensor and held for 2 hours, after which the antibody solution is removed and the concentrations are different. Similarly, five types of elastic wave sensors 30 with antibodies immobilized thereon were prepared. The dropped 20 μl is a volume that allows the solution to reach the entire surface of the sensitive film 22.

  Next, the procedure of the experiment is shown. After 5 minutes from the start of measurement, 20 μl of a reference solution containing no antibody was dropped from the reference solution dropping device 13 onto the reaction field of the measurement acoustic wave element 30 a, and the surface adsorption of antibody molecules was measured by the elastic wave sensor system 10. As shown in FIG. 2, the phase angle does not change before the dropping of the five types of acoustic wave sensors, but the SH-SAW speed changes due to the adsorption of the antibody surface in the solution after the dropping. A change in the phase angle was observed. This phenomenon is similarly reproduced in several reproduction experiments, and the amount of change in phase angle increases as the antibody concentration of the antibody solution increases.

  It was also observed that the phase angle further changed after 10 to 30 minutes from the start of measurement. The present inventor actively removed adsorption other than the antigen-antibody reaction under the assumption that the non-specific adsorption phenomenon of the antibody molecule is affecting the cause of the phase angle further changing over time. The procedure to do was examined.

  FIG. 3 shows an experimental sample of an acoustic wave sensor in which (I) antibody solutions having different concentrations are dropped onto an acoustic wave device for measurement and held for 2 hours, and then the antibody solution is removed and dried in nitrogen (hereinafter ( It is a characteristic diagram showing a comparison between (I) abbreviated) and (II) those obtained by removing the antibody solution and washing the reaction field surface with pure water and then drying with nitrogen (hereinafter abbreviated as (II)). This procedure was added in order to distinguish between changes in properties due to antigen-antibody reaction and changes in properties due to nonspecific adsorption of antibody molecules.

  As shown in FIG. 3, with reference to the value obtained by dropping the reference solution onto the acoustic wave sensor to which the antibody solution is not immobilized, the result of FIG. 3 (I) has less phase change than FIG. 3 (II), and Increases numerical stability. From this, it became clear that highly accurate measurement is possible by eliminating nonspecific adsorption.

  4A to 4C are schematic views schematically illustrating the reason why antibody solutions having different concentrations in this embodiment can be detected as phase angle differences of transverse surface acoustic waves by dropping a reference solution. In the figure, the base portion 31 is made of gold (Au), and a sensitive antigen 32 is provided on the base portion 31, and water molecules of the reference solution 35 are schematically shown around the antigen 32. .

  4A shows a state in which no antibody is present, FIG. 4B shows a state in which a little antibody is immobilized, and FIG. 4C shows a surface of the acoustic wave sensor in which a large amount of antibody is immobilized. Yes. The wavelength of the transverse wave type surface acoustic wave is about 20 μm and propagates in a state where there is no longitudinal wave radiation to the reference solution dropped on the surface and there is little propagation attenuation. Since the shear wave type surface acoustic wave has energy concentrated on the substrate surface, when a substance is fixed on the substrate surface, the sound velocity changes due to a change in the mass of the fixed substance or a change in its viscosity.

  Therefore, the sound velocity of the transverse wave type surface acoustic wave changes and the phase changes depending on the amount of the antibody or antigen immobilized on the base portion 31.

(Second Embodiment)
FIG. 5 shows a configuration diagram of an elastic wave sensor system including an elastic wave sensor according to the second embodiment. In the first embodiment shown in FIG. 1, the measurement acoustic wave element and the reference acoustic wave element are provided. However, in the second embodiment, the measurement acoustic wave is maintained by keeping the temperature during measurement constant. It is possible to measure with the same accuracy using only the element. By adopting such a configuration, it is possible to reduce the size and cost of the acoustic wave sensor.

  As described above, by using this embodiment, the amount of change in the phase angle increases as the antibody concentration of the antibody solution immobilized on the elastic wave sensor increases, and an antigen-antibody reaction can be performed in a simple manner. Can be detected.

It is a block diagram of the elastic wave sensor system containing the elastic wave sensor in 1st Embodiment of this invention. It is the characteristic view which showed the change of the phase angle with respect to time when the phase angle before dripping in the embodiment of the present invention is made into a standard. It is a characteristic figure showing change of a phase angle of 10 minutes after dropping in an embodiment of the present invention. It is a schematic diagram of the antigen antibody reaction in the embodiment of the present invention. It is a block diagram of the elastic wave sensor system containing the elastic wave sensor in 2nd Embodiment of this invention.

Explanation of symbols

  DESCRIPTION OF SYMBOLS 10 Elastic wave sensor system, 11 Signal generator, 12 Phase angle detection circuit, 13 Reference solution dropping device, 14 Comb input electrode, 16 Comb output electrode, 21 Outer wall, 22 Sensitive film, 25 Substrate, 30 Elastic wave sensor, 30a Elastic wave element for measurement, 30b Elastic wave element for reference, 31 Base part, 32 Antigen, 35 Reference solution.

Claims (5)

Changes in the propagation characteristics of surface acoustic waves caused by changes in the measurement sensitive film using a measurement acoustic wave element in which a measurement sensitive film that exhibits adsorptivity to the substance to be measured is formed on a piezoelectric substrate In the detection method for detecting the substance to be measured by
An excitation step of generating a transverse wave type surface acoustic wave by exciting a piezoelectric substrate;
A first phase measuring step for measuring a phase angle before dropping a reference solution, which is a solution for diluting the substance to be measured, on the measurement acoustic wave element on which the substance to be measured is fixed;
A second phase measurement step for measuring a phase angle after dropping the reference solution on the measurement acoustic wave device;
An immobilization amount estimation step for estimating the immobilization amount of the substance to be measured based on the phase angle measured in the first and second phase measurement steps;
Including
A detection method characterized in that the immobilization amount estimation step estimates based on a phase angle change due to a known immobilization amount measured in advance and a phase angle change obtained by measurement. Changes in the propagation characteristics of surface acoustic waves caused by changes in the measurement sensitive film using a measurement acoustic wave element in which a measurement sensitive film that exhibits adsorptivity to the substance to be measured is formed on a piezoelectric substrate In the detection method for detecting the substance to be measured by
The reference acoustic wave element having the same sensitive film as the measurement sensitive film and the measurement acoustic wave element are provided on the same piezoelectric substrate,
An excitation step of generating a transverse wave type surface acoustic wave by exciting a piezoelectric substrate;
A third phase measurement step for measuring the phase angle of both acoustic wave elements when a reference solution, which is a solution for diluting the substance to be measured, is dropped on the acoustic wave element for measurement on which the substance to be measured is fixed;
An immobilization amount estimation step for estimating the immobilization amount of the substance to be measured based on the phase angle measured by both acoustic wave elements measured in the third phase measurement step;
Including
A detection method characterized in that the immobilization amount estimation step estimates based on a phase angle change due to a known immobilization amount measured in advance and a phase angle change obtained by measurement. In an acoustic wave sensor having a measurement acoustic wave element in which a measurement sensitive film showing adsorptivity to a substance to be measured is formed,
An excitation electrode that excites a transverse wave type surface acoustic wave on a piezoelectric substrate, a measurement region in which a measurement sensitive film is formed, and a reception electrode that receives the transverse wave type surface acoustic wave propagated through the measurement region;
An elastic wave sensor comprising an outer wall surrounding a measurement sensitive film on a piezoelectric substrate so that the measurement sensitive film is completely covered with a reference solution which is a solution for diluting a substance to be measured. The elastic wave sensor according to claim 3,
A phase measuring means for measuring a phase angle before dropping the reference solution on the measurement acoustic wave element, and measuring the phase angle after dropping the reference solution on the measurement acoustic wave element;
An immobilization amount estimating means for estimating an immobilization amount of the substance to be measured immobilized on the measurement sensitive film based on the phase angle measured by the phase measurement means;
Have
An elastic wave sensor characterized in that the immobilization amount estimating means estimates based on a phase angle change due to a known immobilization amount measured in advance and a phase angle change obtained by measurement. The elastic wave sensor according to claim 3,
The reference acoustic wave element having the same sensitive film as the measurement sensitive film and the measurement acoustic wave element are provided on the same piezoelectric substrate,
Phase angle measuring means for measuring the phase angle of both acoustic wave elements when the reference solution is dropped onto the acoustic wave element for measurement;
An immobilization amount estimating means for estimating an immobilization amount of a substance to be measured immobilized on a measurement sensitive film based on a phase angle measured by both acoustic wave elements;
Have
An elastic wave sensor characterized in that the immobilization amount estimating means estimates based on a phase angle change due to a known immobilization amount measured in advance and a phase angle change obtained by measurement.

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