JP4156567B2 - SPR sensor and refractive index measuring method - Google Patents

Description

  The present invention relates to a sensor for performing medical diagnosis, environmental analysis, bacteria / virus testing, drug discovery screening, a sensor for detecting an antigen or hormone using an antigen-antibody reaction, a sensor for identifying a DNA sequence, and an enzyme and a substrate. The present invention relates to a surface plasmon resonance (SPR) sensor that measures the concentration of a target molecule using binding, and a refractive index measurement method using the SPR sensor.

(Immunoassay)
Immunoassays that use the antigen molecule recognition function of antibodies are widely used in clinical tests, environmental pollutant measurements, and biochemical measurements because complex biomolecules can be detected without separation. For measurements with relatively low sensitivity, simple measurement methods have been developed and used, for example, for pregnancy tests that can be performed by individuals.

  In addition, since a low concentration of antigen can be measured, it is used for measurement of insulin, BNP (Brain Neutral Peptide), catecholamine, cytokine and other disease markers, environmental hormones, viruses and pathogens that require high sensitivity measurement. Since a specific antibody against a molecule to be measured can be artificially produced at low cost, it is a wide measurement method for target molecules that can measure many natural molecules and non-natural molecules that affect the living body such as environmental hormones.

  Because the binding between the antibody and antigen is very specific and the binding constant is large, the antibody binds to the antigen at a high rate even when the measurement target is at a low concentration, and is not easily affected by contaminant molecules present at a higher concentration. . Due to these characteristics, the immunoassay is a method for measuring a specific molecule with high sensitivity without requiring a separation operation such as liquid chromatography.

(Labeling induced fluorescence method)
When measuring a specific molecule with high sensitivity using the antigen-antibody reaction, the antigen molecule is labeled with a fluorescent molecule, reacted with the antibody immobilized on the substrate, washed, and then the antigen bound to the antibody by induced fluorescence A method for detecting molecules is common. As a method for measuring biomolecules utilizing such specific binding, in addition to the antigen-antibody reaction, there are DNA having a complementary sequence, enzyme and substrate, receptor molecule and ligand molecule. In some cases, a complex of not only one but also a large number of molecules may be formed, and it is measured by labeling with a fluorescent molecule and induced fluorescence as in the immunoassay.

  In order to measure high sensitivity, there are enzyme immunoassay (EIA) and Enzyme Linked Immunosorbent assay (ELISA), but induced fluorescence is a common method for detecting the binding reaction. When a labeling reagent is used, a complicated labeling operation is required (Non-Patent Document 1).

(SPR method)
Therefore, a method for detecting a specific reaction without using a labeling reagent has been developed. Among them, a method of detecting specific binding from refractive index measurement using surface plasmon resonance (SPR) has been put into practical use and widely used (Non-patent Document 2).

  In the measurement method using the SPR method, an antigen or antibody is immobilized on a gold thin film, and when this is specifically bound to a molecule to be measured, the detection is performed by utilizing the fact that the refractive index of the metal thin film surface increases. The SPR method selectively detects changes in the refractive index within a range of several hundreds of nanometers from the surface of a substrate such as gold or silver. If there is a molecular recognition film on the surface of a thin metal film, the refractive index of that thin film is selected. Can be measured automatically. In addition, there is an advantage that even a minute volume can be measured with the same sensitivity as a bulk measurement.

(Simple measurement)
The optical system of the SPR method irradiates monochromatic light onto a high refractive index transparent material on which a gold thin film is formed, and measures total reflected light with a light receiving element. Since strong total reflection light from the light source is measured, an inexpensive light receiving element can be used. It can be realized with a simple optical system, and an inexpensive LED can be used as a light source, and a CCD camera can be used for detection. Because of these features, the SPR method is also suitable for small measuring instruments.

(SPR sensitivity)
In the SPR method, the refractive index is obtained from the incident angle dependence of the P-polarized reflected light intensity from a thin film such as gold. The relationship between the incident angle dependency of the reflected light and the refractive index is uniquely obtained from the Fresnel multilayer film reflectance formula. Therefore, in order to measure high sensitivity, it is necessary to measure the incident angle dependence of P-polarized reflected light intensity with little noise.

  In an apparatus system using the most widely used Kretschmann optical system, wedge-shaped incident light beam, and a two-dimensional imaging device such as a CCD, the minimum reflectance angle (SPR angle) is obtained from the intensity data of the image, and the SPR angle is calculated. Determine the refractive index. In this case, as shown in FIG. 8, the axis representing the incident angle and the axis representing the reflection position on a line perpendicular to the incident surface on the reflecting surface are aligned in the X and Y directions of the pixel, and the reflection intensity becomes the value of the pixel. An image is obtained. In order to perform high-sensitivity measurement, it is necessary to obtain an incident angle at which the reflectance is minimized from this image with high accuracy. However, since there is noise caused by the deviation of the light source, CCD, optical system, and dust, noise also appears at the incident angle at which the reflectance is minimized.

  In order to measure noise reduction, images with different frames are added and averaged over time, or data from reflection points considered to have the same refractive index is added and spatially averaged. One pixel of a CCD or CMOS camera element contains noise, but if this is averaged, extremely stable measurement can be performed. However, the temporal resolution decreases in the former case, and the spatial resolution decreases in the latter case. Also, noise is reduced by measuring the reflected light intensity of S-polarized light having no SPR effect and normalizing P-polarized light. In this case, the optical system becomes complicated.

  However, as shown in FIG. 8, the portion of the image data corresponding to the SPR angle is a dark portion of the image, and it is the dark portion having a small dynamic range that greatly contributes to the determination of the SPR angle. Further, since the SPR angle is translated in the incident angle axis direction of the image as the refractive index changes, it is necessary to measure a portion that does not contribute to the determination of the SPR angle in the angular direction. Furthermore, in order to obtain the minimum reflectance angle with high accuracy using the Fresnel reflection method, it is necessary to measure the absolute value of the reflectance. Not compatible with directional resolution.

(Nanodot SPR)
In addition, a sensor is known that utilizes the effect of shifting the absorption spectrum of a solution containing nano-sized gold fine particles depending on the refractive index of a substance attached to the gold fine particles. Moreover, the same measurement can be performed by forming a silver pattern on the substrate by lithography and measuring the transmission spectrum of the substrate (Non-patent Document 3). In this method, since the refractive index can be measured by measuring the transmission intensity of a specific wavelength instead of measuring the transmission spectrum, it can be used for a simple measuring apparatus.

(Phase detection SPR)
On the other hand, as an SPR measurement method, there is a measurement method using a phenomenon in which a change in refractive index greatly changes the phase of reflected light due to the SPR effect (Non-Patent Documents 4 and 5). In this method, one of the two coherent lights divided in two directions is irradiated onto the gold thin film through a prism, a diffraction grating coupler, or a waveguide with a gold thin film on the core, and the change in light intensity due to interference with the other is detected. The phase difference of the reflected wave from the gold thin film is measured. This method requires an element that divides light with high accuracy and an optical system that multiplexes light. Moreover, it is easily affected by temperature and mechanical deformation, and a special device and structure are required to reduce noise.

(Activity maintenance)
On the other hand, particularly in immunoassay, retention of immobilized antibody activity affects sensitivity and molecular specificity. When immobilized on a substrate, the antibody may lose its activity when immobilized densely and must be dispersed appropriately. For this,
(1) Immobilize antibodies in a polymer three-dimensional network structure formed on a substrate so that the mutual distance increases.
(2) A method of regularly fixing a white self-integrated film formed on a substrate is known.
“Ultrasensitive Enzyme Immunoassay”, Eiji Ishikawa, Academic Publishing Center, published on December 19, 1993, p. 131-132. Biacore, Inc., [online], March 30, 2004 search, Internet URL: http: // www. biacore. co. jp / 3_1_3. shml. A. J. et al. Haes, R .; P. Van Duyne, J.A. Am. Chem. Soc. 2002, 124, 10596-10604. A. Brecht, G.M. Gauglitz, Biosensors & Bioelectronics 10 (1995) 923-936. S. G. Nelson, K .; S. Johnston, S.M. S. Yee, Sensors and Actuators B35-36, (1996) 187-191.

(Low noise measurement)
However, when it is necessary to measure low concentrations of substances that require high refractive index sensitivity or to perform rapid measurements that do not require much time for incubation of the detection reaction at low cost, the noise level can be reduced with a simple device. It is necessary to suppress. In other words, in order to achieve high sensitivity in immunity, DNA hybridization, sensors that use receptor-ligand specific binding reactions, and immunoassays using EIA, ELISA, and Western blotting, the size of the device is complicated. The problem is to reduce the measurement noise without doing so.

(Retention of antibody activity)
Furthermore, when used for molecular recognition by an antibody in SPR measurement, it is necessary to immobilize while maintaining the activity of the antibody. However, in the SPR measurement, since the sensitivity is limited to several hundred nm from the substrate surface, there is a problem that an antibody having a sufficient density cannot always be immobilized when a polymer film is used.

  In order to solve the above problems, an SPR sensor according to the present invention periodically arranges a cluster formed by periodically arranging a plurality of dots of a metal thin film having a surface plasmon resonance effect on a transparent substrate, and The period that is the distance between the centers is longer than the minimum period that is the distance between the centers of the dots.

  In addition, the refractive index measurement method according to the present invention uses the SPR sensor according to claim 1 to bring a substance to be measured into contact with the dot, and irradiates the thin film of the dot with light of a single wavelength. The reflected diffracted light or transmitted diffracted light is observed with a two-dimensional array of light receiving elements, and the refractive index change on the dots is measured from the imaging pattern of the diffracted light from the dots.

As described above, in the SPR sensor and the refractive index measurement method used for the biomolecule interaction measurement according to the present invention,
(1) A stable change in refractive index can be measured with a simple apparatus.
(2) By optimizing the arrangement of dots having a surface plasmon resonance effect, a highly sensitive refractive index can be measured.
(3) It has an effect that the activity of the antibody fixed to the dot having the surface plasmon resonance effect can be maintained.

  The inventors form a repetitive pattern in which a plurality of dots of a thin film having a surface plasmon resonance (SPR) effect are periodically arranged. One unit of this repetitive pattern is defined as a cluster.

  FIG. 1 schematically shows an SPR sensor having such a structure. FIG. 1A shows a single-period structure in which the nano-sized dots 1 are regularly and entirely arranged. That is, the cluster 2 that is one unit of the pattern in which the dots 1 are arranged in a repeating pattern is not provided.

  FIG. 1B shows a double periodic structure, which includes a cluster 2 that is one unit of the pattern in which dots 1 are arranged in a repeating pattern. The cluster 2 has a structure in which the clusters 2 are periodically arranged in a square lattice at predetermined intervals (arranged in a repeating pattern) (see also FIG. 2).

  FIG. 1 (c) shows a triple periodic structure, in which a cluster group 21 in which clusters 2 are arranged in a repeating pattern is formed, and this cluster group 21 is further arranged in a repeating pattern. Yes. FIG. 1 (d) shows multiple cycles, and shows a state in which the clusters 2 are arranged in a plurality of repetitive patterns.

  In this case, the cluster period (distance between centers of clusters; (a + d1) n1-d1 + d2) in FIG. 2) is larger (longer period) than the minimum dot period (distance between centers of dots; d1 + a in FIG. 2). The period of the cluster group (distance between the centers of the cluster groups) is larger (longer period) than the period of. That is, the period increases sequentially from dot to cluster and cluster group (double, triple, and multiple periods).

  The inventors of the present invention have found that a structure having a period of double cycles or more in such a structure can perform stable measurement with extremely high resolution in a measurement method for detecting a specific binding reaction.

  In the measurement method for detecting a specific binding reaction, the present inventors made a material having an SPR effect, in particular, with nano-sized dots arranged in a double cycle, and processed the image of the transmitted or reflected light by image processing. When the refractive index was obtained, it was found that stable measurement with extremely high resolution was possible. More specifically, when combining a periodic pattern (cluster pattern) for diffracting incident light and a dot pattern of nano-sized dots to efficiently immobilize molecules such as antibodies, highly sensitive refraction It was possible to measure the rate, and found that the antibody was immobilized while maintaining high activity, and reached the present invention.

  A typical structure according to the present invention is a double periodic structure as described above. Referring to FIG. 2 to be described later, the dots 1 of the metal thin film exhibiting the SPR effect are formed in a square lattice at predetermined intervals (in a repeating pattern or periodically arranged). Preferably, the minimum period (distance between the centers of dots; d1 + a in FIG. 2) of this repetitive pattern is 30 to 110 nm. More preferably, it is 80-110 nm. As shown in Example 1, if the thickness is less than 30 μm, it is difficult to produce, while if the minimum period exceeds 110 nm, the distance between dots becomes long and the SPR effect becomes weak.

  Further, the maximum width of dots (in the case of a square as shown in FIG. 2, the length of one side; the maximum outer diameter in the case of a circular shape, a star shape, etc.) a is preferably 60 nm or less. As is clear from Example 2, when the thickness is 60 nm or less, the recovery of the diffraction pattern by repetition is good, and it is understood that this condition is a preferable condition for maintaining the activity of the antibody.

  Furthermore, the cluster interval d2 is preferably 1600 nm or less and 400 nm or more. This is because the diffraction intensity is large at 1600 nm or less, and the contrast of the image is increased. Further, if it is less than 400 nm, the number of minimum period patterns constituting the cluster is reduced (because the minimum dot period is smaller than the cluster period). That is, there is a risk that the double periodicity is lost.

  In the present invention, it is possible to modify a membrane containing a biomolecule that causes a specific binding reaction with a dot that exhibits the SPR effect. For example, anti-IgG can be modified through a self-assembled membrane terminated with a carboxyl group on a gold dot, or it can be modified directly with a sulfide group using F (ab ′) 2 or Fab fragments. it can.

  Furthermore, in the conventional SPR measurement, the incident angle dependence of the reflected light by SPR is measured using a part of the reflected light beam.

  On the other hand, in the present invention, a substance to be measured is brought into contact with the dot, the thin film of the dot is irradiated with light having a single wavelength, and reflected or transmitted diffracted light from the dot is two-dimensionally arrayed. Observation with a light receiving element, the refractive index change on the dot is measured from the imaging pattern of the diffracted light from the dot.

  By using the diffraction image due to the phase change of the reflected or transmitted light by the SPR, the refractive index information of the object to be measured is reflected in the entire diffraction image, has a large averaging effect, and can be measured with low noise even with an inexpensive device. High sensitivity can be achieved.

  Hereinafter, the present invention will be described in detail with reference to the drawings. In addition, this invention is not limited only to a following example.

  As shown in FIG. 2, a gold thin film dot having a thickness of 45 nm and a maximum width (in the case of a square as shown in FIG. 2, the length of one side; the maximum outer diameter is shown in the case of a circular shape, star shape, etc.) On the transparent glass substrate 3 of BK7, a cluster 2 in which n1 × n1 clusters 1 are arranged in a square lattice at a spacing d1 nm and a square lattice in a spacing d2 nm is formed on a transparent glass substrate 3 of BK7. Various combinations of a, d1, d2, and n1 were prepared. This SPR sensor S was produced by electron beam lithography and dry etching. The occupied part of all the clusters 2 is about 1.4 × 1.4 mm. First, when the transmission spectrum of this gold dot substrate (SPR sensor) was measured, it was found that absorption appeared at 575 nm, and that the gold dot 1 exhibited a surface plasmon resonance effect.

  Next, the SPR sensor S was irradiated with a laser having a wavelength of 670 nm from the semiconductor laser 4 with a measuring apparatus as shown in FIG. 3, and a diffraction image was taken with the CCD camera 5 on the reflection side to obtain the image of FIG. The vertical stripes in FIG. 4 are obtained by diffracting the cluster 2 of gold dots 1 in contact with air in the direction of the camera. This diffraction pattern (imaging pattern) corresponds to the refractive index of air (1.0004).

  Next, the diffraction pattern was measured in the same manner while the pattern surface of the gold dot 1 was in contact with water, and another diffraction pattern was obtained. Since the refractive index of water is 1.33, it can be seen that this refractive index difference gives a large change to the diffraction pattern. In this case, since the distance between the vertical stripes of the diffraction pattern and the position move with the change of the refractive index, the material contacting the gold dot 1 from the diffraction pattern by a method such as calculating the average distance and the center of gravity of the diffraction pattern. Can be measured.

  As shown in FIG. 4, the refractive index information on the gold dot 1 due to the SPR phenomenon appears in the entire image, and when this image is temporally added, it has been found that the information of one pixel takes a stable value. Also, when calculating the fringe period and position, all the pixels have the same weight, so that all the information of the image can be used efficiently to determine the refractive index.

  Next, change the a, d1, d2, and n1 that determine the arrangement of the square lattice pattern of the dots 1 and clusters 2, and change the time for producing the pattern, the yield of the SPR sensor chip, and a laser with a wavelength that can be obtained at low cost It was investigated whether a diffraction pattern changing with refractive index change could be obtained.

  The result is shown in FIG. As is apparent from FIG. 5, the minimum period (dot center distance) of the dot 1 is 30 nm or more, preferably 80 nm or more, from the viewpoint of ease of production. If it is less than 30 nm, it is difficult to form a stable dot pattern and the dot pattern yield is low. A thickness of 80 nm or more could be stably produced.

  On the other hand, with respect to the maximum value of the minimum period, when the minimum period exceeded 110 nm, the distance between dots increased and the SPR effect was weakened. Moreover, when the area occupied by the dots was increased, the activity maintaining effect of the antibody was weakened, which was not suitable.

  The cluster interval d2 (period is (a + d1) n1-d1 + d2)) is 1600 nm or less, preferably 800 nm or less, at which the diffraction intensity is large and the image contrast is increased. A sufficiently large contrast was obtained at 800 nm or less. On the other hand, when the cluster period is shortened, the number of period patterns constituting the cluster is small, and the double periodicity is lost. Therefore, the period must be 400 nm or more.

  In order to increase the amount of change, the change in the diffraction pattern can be designed in advance, and the arrangement of the gold dots may be a periodic arrangement other than a square lattice pattern. Any shape can be used for the dot shape.

  As shown in FIG. 6, a self-assembled film terminated with a carboxyl group on gold was prepared on the gold dot 1 of Example 1, and an anti-IgG manufactured by Sigma was immobilized thereon with a covalent bond (specific binding). Membrane containing biomolecules that cause a reaction). In the antibody immobilization method, F (ab ') 2 or Fab fragment may be used and directly immobilized with a sulfide group. Next, the gold dot chip (SPR sensor) S modified with anti-IgG was adhered to the microchannel 6 so that the gold dot pattern surface 11 on which the gold dots 1 were formed, as shown in FIG. The flow path 6 has a flow path height of 0.02 mm and has a through hole 7.

  Next, the micro flow channel 6 with a gold dot chip (SPR sensor) and the glass substrate 8 with the introduction and discharge groove 81 opened were bonded. Pipes were connected to the liquid supply port 82 and the discharge port 83, and liquid was supplied from the liquid supply port 82 using a syringe pump. The liquid passes through the groove 81 from the liquid supply port 82, enters the microchannel 6 through the hole 7, contacts the gold dot pattern surface 11 of the SPR sensor S, and is discharged from the discharge port 83 through the hole 7 and groove 81. It has become so. The SPR sensor S is irradiated with a laser beam from a semiconductor laser, and a diffraction image is taken on the reflection side of the CCD camera 5 to measure diffraction.

  First, a phosphoric acid buffer containing bovine serum albumin (BSA) manufactured by Sigma was flowed, and the diffraction from the gold dot pattern 11 was measured while feeding so that the phosphoric acid buffer was in contact with the gold dot pattern surface 11.

  Next, when a solution containing IgG produced by Sigma, which is an antigen against anti-IgG, is sent, the interval and position of the diffraction pattern change continuously, and the solution of IgG is stopped. When switched to liquid feeding, the diffraction pattern became constant.

  Furthermore, when a glycine buffer having pH 5 was fed and then a phosphate buffer containing BSA was fed, the initial diffraction pattern was recovered. When this experiment was repeated for gold dot patterns having different values of a, it was found that when a was 60 nm or less, the recovery of the diffraction pattern was good by repetition, and this condition was a favorable condition for maintaining antibody activity. It was. In addition to the antigen-antibody reaction such as anti-IgG and IgG, this experiment works similarly with complementary single-stranded DNAs or DNA-RNA combinations, and can be used as a DNA sensor.

In the present invention, when detecting a specific binding reaction, an antibody or other molecule is immobilized on a nano-sized dot in which a material having an SPR effect is arranged in a double cycle, and an image of the transmitted light or reflected light is image-processed. Then, the concentration of the target molecule is measured by measuring the refractive index. For this reason,
(1) A stable change in refractive index can be measured with a simple apparatus.
(2) By optimizing the arrangement of dots having a surface plasmon resonance effect, a highly sensitive refractive index can be measured.
(3) There is an advantage that the activity of the antibody fixed to the dot having the surface plasmon resonance effect can be maintained.

Explanatory drawing explaining the period of the dot and cluster of this invention. Explanatory drawing explaining the period of the dot and cluster of the Example of this invention. Schematic explaining the measurement system of the Example of this invention. The figure which shows the diffraction pattern obtained in the Example. The figure which shows the relationship between the minimum period of a dot of an Example, and a cluster space | interval. FIG. 5 is an explanatory diagram of an SPR sensor used in Example 2. Explanatory drawing of the measurement system used in the Example. The figure which shows the conventional diffraction pattern.

Explanation of symbols

1 dot 11 gold dot pattern surface 2 cluster 21 cluster group 3 transparent substrate 4 semiconductor laser 5 CCD camera 6 micro flow path 7 hole 8 glass substrate 81 groove 82 liquid supply port 83 discharge port

Claims (10)

A cluster formed by periodically arranging a plurality of dots of a metal thin film having a surface plasmon resonance effect is periodically arranged on a transparent substrate, and a period which is a distance between the centers of the clusters is a distance between the centers of the dots. An SPR sensor having a longer period than a certain minimum period. The SPR sensor according to claim 1, wherein the clusters in which the plurality of dots are arranged in a square lattice at a predetermined cycle are arranged on the transparent substrate in a square lattice at a predetermined cycle. The SPR sensor according to claim 2, wherein a minimum period of the dots is 30 to 110 nm. 4. The SPR sensor according to claim 3, wherein the minimum period of the dots is 80 to 110 nm. The SPR sensor according to any one of claims 2 to 4, wherein an interval between the clusters is 400 to 1600 nm. The SPR sensor according to claim 5, wherein an interval between the clusters is 400 to 800 nm. The SPR sensor according to any one of claims 2 to 6, wherein the maximum width of the dots is 60 nm or less. The SPR sensor according to any one of claims 2 to 7, wherein all or part of the dots are modified with a film containing a biomolecule that causes a specific binding reaction. The SPR sensor according to claim 1 is used, the substance to be measured is brought into contact with the dot, light of a single wavelength is irradiated on the thin film of the dot, and reflected or transmitted diffracted light from the dot is two-dimensionally emitted. A method for measuring a refractive index, comprising: observing with an arrayed light receiving element, and measuring a change in refractive index on the dot from an imaging pattern of diffracted light from the dot. A method of measuring a refractive index, wherein the SPR sensor according to claim 8 is used to measure a refractive index change caused by a molecule binding specifically to the film.

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