JP2005069980A - Surface plasmon resonance system - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a surface plasmon resonance system of high performance capable of measuring many samples with excellent reproducibility and reduced dispersion, by obtaining a light beam reduced in illuminance unevenness. <P>SOLUTION: In this surface plasmon resonance system, an area of a measuring area is 70 mm<SP>2</SP>or more when 1% of aqueous ethanol solution is brought into contact with a surface of a sensor to be measured, using water as a base line, in the case where a rectangular or square measuring area having 0.5 or more of aspect ratio is provided within a sensor surface portion of the surface plasmon resonance system, where the measuring area is divided evenly into vertical ten pieces ×lateral ten pieces of divided measuring areas, and where a circular marked area having a diameter corresponding to 40 % of short side in the divided area is provided in the center of the divided measuring areas. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

  The present invention relates to a surface plasmon resonance apparatus in which variation in signal is reduced because there is little unevenness in illuminance of a polarized light beam applied to a thin metal layer.

  In recent years, many attempts have been made to evaluate the interaction between physiologically active substances. Many observe the interaction by immobilizing one molecule on a solid phase and providing a substance that will interact with that molecule. Labeling substances such as fluorescence and radioisotopes are bound to substances that will interact, and the label substances are detected to evaluate interactions between biomolecules or molecular assemblies. A general DNA chip screens an interaction using a label substance such as fluorescence. However, label operations are very complicated and lack quantitativeness. Also, it is very difficult to observe the interaction in real time.

  Therefore, a surface plasmon resonance (SPR) sensor has attracted attention as an analysis method that does not require a label, has quantitativeness, and can be evaluated in real time. SPR is a method in which reflected light is monitored by irradiating a metal thin film on which an analyte is fixed, and the interaction with a sample is measured by a change in resonance angle or reflected light intensity.

  In a general SPR sensor, laser light having a single wavelength is used as a light source (see, for example, Patent Document 1). Laser light is excellent in straightness, and is a very easy light source for optical research. However, light is a concentrated beam, and the area of the light hit is very small. Therefore, there is basically one substance that can evaluate the interaction, and it is very difficult to screen a plurality of substances such as a DNA chip.

  As a means for solving this problem, it is conceivable to apply a method (for example, see Patent Document 2) in which laser light is reflected and scanned by a scanning device and laser light is sequentially irradiated onto the surface of the slide. However, there is a problem that real-time evaluation cannot be performed while the laser beam is scanned.

  Accordingly, Nelson et al. Selected a 35 W halogen lamp as the SPR light source. The light was collimated and then collected in a pin hole having a diameter of 300 μm, and was again collimated by the camera lens. The obtained parallel light is passed through a polarizing plate, incident on the SPR at an appropriate angle, irradiated with a gold-deposited slide of 18 × 18 mm, and the reflected light is passed through a wavelength filter before being photographed with a CCD camera (for example, non- Patent Document 1).

The data shown here is only for 24 spots in a 6 mm × 4 mm = 24 mm ² area on the sensor surface. Although it has been shown that there is almost no variation with respect to the 24 data, it is difficult to obtain a uniform signal even if spots are provided over a wider range than that.

  As a means for obtaining a uniform signal over a wide range, it is necessary to obtain uniform illuminance. Although there is a method to reduce the pinhole hole as a means to increase the illuminance uniformity, sufficient illuminance cannot be obtained, and the signal uniformity may be reduced. There was a problem that required a camera.

European Patent Application No. 0341927 International Publication No. 93/14393 Pamphlet Nelson et al., Anal.Chem., 71, 3928-3934, 1999

  An object of the present invention is to provide an SPR having a high performance in which a large number of samples can be measured with good reproducibility and with little variation by obtaining a light flux with reduced illuminance unevenness.

  As a result of intensive studies, the present inventors have found that the above problems can be solved by the following means.

1. A rectangular or square measurement area having an aspect ratio of 0.5 or more is taken in the sensor surface portion of the surface plasmon resonance apparatus, and the measurement area is equally divided into 10 vertical measurement areas and 10 horizontal measurement area areas. When a circular region of interest having a diameter corresponding to 40% of the short side of the divided region is provided at the center of the substrate, water is used as a baseline and measurement is performed with a 1% ethanol aqueous solution in contact with the sensor surface. A surface plasmon resonance apparatus characterized in that an area of a measurement region in which variation (CV) of a signal obtained from each region of interest can be 15% or less is 70 mm ² or more.
2. 2. The apparatus according to 1, wherein the diameter of the pinhole used for homogenizing the light source is 50 μm or more and less than 300 μm. 3. The apparatus according to 1 or 2, wherein a square filament type halogen lamp is used.

  By obtaining a polarized light flux with high illuminance and no illuminance unevenness, an inexpensive SPR device with little signal variation was realized. In the future, it is expected to be widely used for screening biomolecule interactions.

  The present invention is described in detail below. The SPR device of the present invention is characterized in that it can irradiate polarized light flux having sufficient illuminance and no illuminance unevenness over a wide area of the chip in order to analyze a wide range of SPR signal changes.

The wide range referred to here is an area on the chip surface of 70 mm ² or more. That is, means for irradiating a narrow area with laser light or means for condensing the light source and irradiating a point or line on the sensor are not included. If it is less than 70 mm ² , the area is not sufficient, and it becomes difficult to secure the n number of screening tests and observe the morphology.

  As a method for quantitatively measuring the signal unevenness of the SPR device of the present invention, a test chip is set on the device, two kinds of liquids having different refractive indexes are sequentially flowed, and the signal change of each part obtained by dividing the observation region is measured. A method of quantifying variation is preferred. It is not preferable to directly measure the illuminance unevenness of the polarized light flux because it does not reflect the SPR performance. However, it is not preferable to simply measure the variation in the illuminance of the reflected image because minute dirt on the chip cannot be distinguished from the variation in illuminance.

As two types of liquids having different refractive indexes, water and a 1% ethanol aqueous solution are preferable because they are easily available. The measurement area is preferably a wide area of 70 mm ² or more, more preferably 80 mm ² in terms of the surface of the test chip.
The measurement area is preferably a rectangle or square having an aspect ratio of 0.5 or more. As an aspect-ratio, More preferably, it is 0.6 or more, More preferably, it is 0.7 or more. If the aspect ratio is lower than 0.5, the measurement area becomes too long and becomes substantially difficult to use. Here, the aspect ratio of OO or more means that the numerical value is larger than OO.
When it is less than 70 mm ^2, it is difficult to accurately evaluate many samples at once. This is because even if there is little variation in a narrow region, it is meaningless if there is variation in a wide region.

The variation in signal change in the measurement area is measured as follows.
The measurement area is equally divided into 10 vertical areas and 10 horizontal areas, and each area is defined as a divided area. Further, a circular measurement point (region of interest) for measurement is provided at the center of the divided measurement region, and the signal in this region is measured by SPR. The diameter of the attention area corresponds to 40% of the length of the shorter side of the vertical and horizontal sides of the divided measurement area. A signal is detected in each region of interest, the difference between the signals of water and a 1% aqueous ethanol solution is determined, and the average value and standard deviation of the signal change in these 100 regions of interest are determined.

Thus, the variation in the change in signal obtained with water and 1% aqueous ethanol is indicated by CV. CV is a value obtained by dividing the standard deviation by the average value. The CV value is preferably 15% or less, and more preferably 10% or less.
By using an SPR measuring instrument in which such a CV value measurement region is 70 mm ² or more on the sensor surface, many samples can be measured simultaneously and accurately.

  The test chip is preferably a transparent substrate coated with gold. The thickness of gold is preferably 30 to 100 nm, and particularly preferably 40 to 60 nm. In order to prevent gold peeling, chromium or titanium may be coated with 1 to 10 nm between gold and the transparent substrate. The metal coating method is not particularly limited, and examples thereof include a vacuum deposition method, a sputtering method, and an ion coating method. In actual use, it is preferable to coat alkanethiol with a single molecule on the gold surface because the chip surface is not soiled. As the alkanethiol, it is preferable to select 11-mercaptoundecanol having an OH group, 11-mercaptoundecanoic acid having a COOH group, or the like.

  The transparent substrate is not particularly limited, and examples thereof include glass and plastics. A flat substrate or a diffraction grating substrate is also included.

  A general inexpensive light source has a filament portion, and there is unevenness in light brightness. If the light from the light source is irradiated as it is, bright and dark unevenness occurs in the image obtained by reflection, making it difficult to evaluate screening and morphological changes. As a means for uniformly irradiating the chip, a method in which light passes through a pinhole and then becomes parallel light is preferable.

  The means for passing a pinhole is preferable as a means for obtaining a light beam having a uniform brightness, but there is a drawback that the illuminance decreases when light is passed through the pinhole as it is. Therefore, as a means for ensuring sufficient illuminance, it is preferable to use a method in which a convex lens is installed between the pinhole and the light source, and condensed to pass through the pinhole.

  The shape of the pinhole is not particularly limited to a shape such as a circle, an ellipse, a quadrangle, a polygon more than that, or an indefinite shape, but a circle is preferable.

  The size of the pinhole is preferably 50 μm or more and less than 300 μm in diameter. Furthermore, 100 micrometers or more are preferable. If it is less than 50 μm, it is not preferable because sufficient illuminance cannot be secured. When a pinhole of 300 μm or more is used, or when a pinhole is not used, the illuminance unevenness of the light beam occurs, and an accurate imaging analysis may not be possible as an SPR device. Here, the diameter means the diameter when the pinhole is circular, the diameter of the long axis when it is an ellipse, and the longest value on a diagonal line when it is a polygon.

  The light that intersects and passes through the pinhole is preferably converted into parallel light by using a condensing lens (convex lens), transmitted through the polarizing plate, and then irradiated onto the sensor surface.

  The light source used in the present invention is preferably an inexpensive halogen lamp. Among them, a halogen lamp having a square filament is very preferable because unevenness in illuminance is reduced. The lamp life is preferably 1500 hours or more, and the total luminous flux representing the brightness of the lamp is preferably 300 lumens or more. As a method for guiding light, a method using an optical fiber is also possible.

  In this way, for example, a surface plasmon resonance device in which variations in illuminance are reduced by using a halogen lamp having a pinhole with a small diameter and a square filament shape can be realized.

  The illuminance of the polarized light beam obtained as a result is preferably 20 lux or more. If it is 20 lux or more, even after the SPR reflection image passes through the wavelength filter, it can be taken with a commercially available general CCD camera. However, a simple and inexpensive white light source is used as a light source, and light is passed through a pinhole in order to uniformly irradiate a polarized light beam. Therefore, it is expensive to increase the illuminance, and 500 lux or less is preferable. .

  The reflected light from the chip passes through a wavelength filter and is photographed by a CCD camera, and only light in the vicinity of a specific wavelength is observed. The center wavelength of the wavelength filter is not particularly limited, but 650 nm to 1000 nm having high SPR sensitivity is selected. When the center wavelength of 750 to 950 nm is selected, the SPR becomes more sensitive. The wavelength width of the wavelength at which the transmittance of the wavelength filter is half that at the maximum is called a half-value width. A smaller half-value width is preferable because the wavelength distribution is sharper. Specifically, a half-value width of 50 nm or less is preferable, and a half-value width of 15 nm or less is particularly preferable.

  An image photographed by the CCD camera is taken into a computer, and a change in brightness of a certain part can be evaluated in real time, or an entire image can be evaluated by image processing. Thus, it is possible to screen a chip on which a plurality of substances are immobilized, and to observe the morphology of an object adsorbed on the surface with high sensitivity.

  The surface plasmon resonance apparatus according to the present invention is a sensor having very high performance with little illuminance unevenness, and is suitably used for various analyses.

  EXAMPLES The present invention will be specifically described below with reference to examples, but the present invention is not limited to the examples.

[Example 1]
A square filament type halogen lamp (manufactured by Hosobu Inc.) was used as the light source. Although this lamp has a high brightness of 30 W and a total luminous flux of 440 lumens, it has an average life of 2000 hours and can be used for analytical instruments.

A spherical plano-convex lens (SLSQ30-50P manufactured by Sigma Koki Co., Ltd.) having a diameter of 30 mmφ and a focal length of 50 mm was placed at a position 50 mm from the light source with the plane side of the lens facing the lamp, and the light from the light source was made parallel light. A further 35 mm away from the spherical plano-convex lens, another spherical plano-convex lens (SLSQ30-50P manufactured by Sigma Koki Co., Ltd.) having a diameter of 30 mmφ and a focal length of 50 mm was placed facing the light source. The spherical plano-convex lens collects light at a position of 50 mm from the lens. A pin hole (PA-200 manufactured by Sigma Kogyo Co., Ltd.) having a diameter of 200 μm is placed at that position, and the light beam is passed through the pin hole to obtain a spherical aromatic lens (Sigma light). Machined DLB-30-50PM), and a parallel light beam having a diameter of 24 mm was obtained with a near-infrared polarizing filter (SPFN-30C-26 made by Sigma Koki Co., Ltd.) having an effective diameter of 24 mm. The cross-sectional area of this polarized parallel light beam is 452 mm ² and can irradiate a wide area. The brightness was 60 lux and the brightness required for the present invention when measured with a digital illuminometer LM-331 having a photosensitive area of 10 mmφ.

Self-assembled 11-mercaptoundecanol on a gold surface on which 3 nm chromium and 45 nm gold are deposited on a Lak10 slide (n _D = 1.720, manufactured by Matsunami Glass Industrial Co., Ltd.) 18 mm square and 1 mm thick Slides on which molecular layers (SAMs) were formed were used as test chips. Matching oil (Cargill M series, nD = 1.720) is applied to the glass surface of the test chip and brought into contact with a 25 ° side 60 ° dispersive prism (Oriel 46106), and the parallel polarization of the SPR device of the present invention. Introduced luminous flux. Water was brought into contact with the SAMs surface of the test chip.

  The reflected light beam from gold is extracted at a wavelength near 830 nm by an optical interference filter of 830 nm (VPF-25C-10-45-83000 manufactured by Sigma Kogyo Co., Ltd.). After taking out only parallel light, it was photographed with a black and white CCD camera (CS8330B manufactured by Tokyo Electronics Industry Co., Ltd.). The outline of the apparatus is shown in FIG. This CCD camera is relatively inexpensive and the minimum subject illumination is 0.3 lux. It was possible to detect an image by SPR measurement.

In order to evaluate the variation of the signal, water was allowed to flow through the SAMs surface of the test chip at a rate of 100 μl / min, and images were taken about every 5 seconds. While photographing was continued, a 1% ethanol aqueous solution was introduced onto the surface of the SAMs, and a change in SPR signal due to a change in refractive index when water was again flowed was observed. The observation was performed on a portion (measurement region) corresponding to a region of 10 mm square (= 100 mm ² ) near the center of the SAMs surface. The area was divided into 100 equal parts (10 × 10), and 100 attention areas corresponding to a diameter of about 400 μm were created at the center of each divided measurement area, and the change in signal was measured. The signal change by 1% ethanol at each point is shown in FIG.

  The average signal of water and 1% ethanol in 100 regions of interest was 16.9, the standard deviation was 1.26, and the CV was 7.5%. An SPR device with reduced signal variation could be obtained.

[Comparative example]
A general-purpose halogen lamp (Philips 13078) was used as the light source. This lamp is 20W. Using this lamp, the pinhole of the apparatus of the above example was exchanged for a diameter of 300 μm (PA-300 manufactured by Sigma Koki Co., Ltd.), and the same measurement was performed. The measurement results are shown in FIG. The signal average of 100 regions of interest was 15.8, which was the same as that of the example, but the standard deviation was 2.95, and the CV was 18.7%, which was a very bad result. Further, although the measurement area was narrowed, an area where the CV value could be 15% or less could not be secured at 70 mm ² or more.

  By obtaining a polarized light flux with high illuminance and no illuminance unevenness, an inexpensive SPR device with little signal variation was realized. In the future, it is expected to be widely used for screening biomolecular interactions, and it will greatly contribute to the industry.

Overview of SPR imaging device Changes in SPR signal by 1% ethanol in the examples Change of SPR signal by 1% ethanol in the comparative example

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 Square filament type halogen lamp 2 CCD camera 3 CCTV lens 4 Flow cell 5 Pinhole 6 Near-infrared polarizing filter 7 Interference filter (830 nm)
8 60 degree prism 9 Plano-convex lens 10 Plano-convex lens

Claims (3)

A rectangular or square measurement area having an aspect ratio of 0.5 or more is taken in the sensor surface portion of the surface plasmon resonance apparatus, and the measurement area is equally divided into 10 vertical measurement areas and 10 horizontal measurement area areas. When a circular region of interest having a diameter corresponding to 40% of the short side of the divided region is provided at the center of the substrate, water is used as a baseline and measurement is performed with a 1% ethanol aqueous solution in contact with the sensor surface. A surface plasmon resonance apparatus characterized in that an area of a measurement region in which variation (CV) of a signal obtained from each region of interest can be 15% or less is 70 mm ² or more.   2. The apparatus according to claim 1, wherein the diameter of the pinhole used for homogenizing the light source is 50 μm or more and less than 300 μm. 3. The apparatus according to claim 1, wherein a square filament type halogen lamp is used.