JP3900294B2 - Surface plasmon resonance device - Google Patents

Description

  The present invention relates to a two-dimensional surface plasmon resonance apparatus with reduced distortion and distortion and easy mask operation.

  In recent years, many attempts have been made to evaluate the interaction between physiologically active substances. In many cases, one molecule is immobilized on a solid phase, a label substance such as fluorescence or radioisotope is bound to the molecule and the analyte (analyte), and the label substance is detected to detect between the biomolecule or molecular assembly. A method has been taken to evaluate the interaction. 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 a metal thin film on which an analyte is immobilized is irradiated with light to drive reflected light, and the interaction with the 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 as seen in 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 to solve this problem, Nelson et al. Adopted a halogen lamp as a light source of SPR, made the light source parallel light with a spatial filter combining a convex lens and a pinhole, passed through a polarizing plate, and suitable for SPR. Simultaneously measure multiple substances with a so-called two-dimensional surface plasmon resonance device, which is incident at an angle, irradiates a gold-deposited slide of 18 x 18 mm, and passes the reflected light through a wavelength filter and then images with a CCD camera. (Non-Patent Document 1).

  However, in the prior art, since the camera lens used has a single lens configuration of a general CCTV lens or a convex lens, two-dimensional imaging itself is possible, but spherical aberration, distortion, and image accompanying lens aberration are possible. The target two-dimensional SPR image, such as distortion and defocusing of the image due to the so-called perspective effect due to distortion and defocus due to surface curvature, etc. Distortion is large and blurry.

Further, in such a two-dimensional surface plasmon resonance apparatus, a substance that interacts with the analyte is immobilized on a predetermined region on the gold thin film, and spots are formed. Therefore, each SPR signal is measured. In this case, it is necessary to perform a so-called mask operation for designating an area to be observed for each substance to be measured based on an image taken before measurement. Usually, the spots have the same shape, the same area, and are arranged at equal intervals. However, as described above, in the conventional technique, a photographed image has a large distortion, and the original pattern cannot be faithfully reproduced. For this reason, the mask operation cannot be specified collectively by equal interval processing, and it is necessary to specify all the areas one by one, which is very complicated when the number of spots is large.
EP0341927Al WO93 / 14393 Nelson et. a1. Anal, Chem. 1999, 71, 3928-3934

  An object of the present invention is to reduce image distortion in a two-dimensional surface plasmon resonance apparatus, thereby enabling simplification by equally dividing batch designation of mask operation, reduction of SPR signal variation, high-density SPR simultaneous measurement, and the like. That is.

As a result of intensive studies, the present inventors have found that the above problems can be solved by the following means.
1. A measuring apparatus using surface plasmon resonance, having a spot for measuring a sample on a chip and having a light receiving means for measuring reflected light from a metal thin film surface on the chip in two dimensions. A telecentric lens is used, and the working distance (WD) of the telecentric lens is in the range of 50 to 200 mm, and the region where the distortion of the reflected image is 4% or less is continuously at least 25 mm ^{2 to} 10,000 mm on the chip. A two-dimensional surface plasmon resonance apparatus having ^two or less.
2. The apparatus of 1 whose absolute value of the distortion in the vertical direction and the distortion in the horizontal direction are both 0.5% or less.
3. 1 or 2 apparatus, wherein the number of simultaneously measurable samples on the chip is 25 or more.
4). 3. The apparatus according to 3, wherein the number of simultaneously measurable samples on the chip is 25 or more and 27889 or less.
5 . A measurement method comprising the step of simultaneously measuring 25 or more spots on a chip using one or two two-dimensional surface plasmon resonance apparatus.
6 . A measurement method comprising the step of simultaneously measuring 25 or more and 27889 or less spots on a chip using one or two two-dimensional surface plasmon resonance apparatus.

  The array that is the subject of the present invention is an array of a large number of spots. The number of spots is 5 or more, preferably 6 or more, more preferably 7 or more on at least one side. Further, even in a side with few spots, the number of spots is 4 or more, preferably 5 or more, more preferably 6 or more.

The array of the present invention usually has an area of about 25 mm ² or more in the measurement region. The size of about 25 mm ² is assumed to be about 5 mm × about 5 mm, for example. Assuming that the size of one spot on the array is 0.3 to 0.5 mm and the interval is also 0.3 to 0.5 mm, 25 spots (5 × 5 pieces) on a measurement area of about 25 mm ^2. Measurement is possible at spots of about 0.5 spot (0.5 mm, interval 0.5 mm) to about 50 or more (7 × 7-8 × 8; spot 0.5 mm, interval 0.5 mm). In the case of simultaneously measuring many spots as described above, if the distortion of the reflected image is 4% or less, it is possible to realize masking at equal intervals and facilitate processing.

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

  As shown in FIG. 3, the two-dimensional plasmon resonance device is configured to first enter a p-wave modulated parallel light beam. The parallel accuracy of the parallel light and the uniformity of the illuminance in the light beam are the performance of the SPR. Is important. Therefore, it is preferable to use a halogen lamp as a light source and a so-called spatial filter using a convex lens and a pinhole in the middle.

  Next, the metal thin film surface and the substance to be measured are brought into contact in two dimensions. An optical prism and an optical slide glass having the same refractive index as that of the optical prism coated with the metal thin film are subjected to the same refraction. It is connected to the opposite side of the gold thin film surface through a matching oil having a rate, and has a recess such that a two-dimensional liquid space is provided between the metal thin film surface and connected to the liquid space. A method of joining a flow cell having an input / output port and an optical slide glass (Kretschmann arrangement) is preferable. However, a method in which a diffraction grating is provided on the metal surface and no prism or matching oil is required (Wood arrangement), and a method in which a gap is provided between the prism bottom surface and the metal surface (Ototo arrangement) are also possible.

  As a means for measuring the two-dimensional reflected light intensity used in the present invention, a television camera is preferable. Further, as a type of camera, any camera tube, CCD, CMOS camera can be used as long as a single color can be photographed two-dimensionally. However, a CCD camera is more preferable in terms of both economy and performance. The camera size and the number of pixels are not particularly limited as long as the resolution including the lens is equal to or higher than the resolution of the target metal thin film surface.

In the present invention, the distortion of the two-dimensional reflected light, that is, the reflected image is preferably 4% or less, more preferably 3% or less, and particularly preferably 2% or less. This is because the mask operation is easier when the distortion is smaller. The distortion is calculated by measuring the distance of the location shown in FIG. 5 from the reflection image from a square area of 25 mm ² or more on the chip.
Strain (%) = (| L _b −L _u | / L _b ) × 100 Formula (1)
L _u : Length from the left end of the leftmost spot to the right end of the rightmost spot on the top row L _b : Length from the left end of the leftmost spot to the right end of the rightmost spot of the bottom row The distortion of the reflected image on the chip is 4% If the area to be described below is less than 25 mm ² , the area where the spots are arranged as an array becomes narrow, and there are cases where the merit of the two-dimensional surface plasmon resonance measurement that many samples can be measured at one time cannot be fully utilized. Since the optical system requires an incident angle that causes the SPR phenomenon, the reflected image is taken from an oblique direction, not from the front of the chip. As a result, the image has a shape close to a rectangle, but the aspect ratio is not limited in the present invention.

  The meaning of distortion of 4% or less will be described below by taking a case of a region of 10 mm × 10 mm as an example in order to facilitate calculation.

When the strain is 4% or more, assuming an area of 10 mm and 10 mm (100 mm ² ), the upper side is 10 mm and the lower side is 9.6 mm (or vice versa). In this case, a deviation of 0.4 mm occurs.

  Assuming that the size of one spot on the chip is 0.5 mm in diameter or 0.5 mm on a side (if the spot is larger than this, it is difficult to obtain a sufficient number of measurable spots on the chip), it is caused by distortion. When the center of the spot is shifted by 0.2 mm, the overlapping portion of the spot becomes 0.3 mm, which is a measurable limit, and can be measured only when a very small mask is used.

  Depending on the size of one spot on the chip, the distance between spots, symmetry of distortion, etc., the allowable distortion ratio changes, but if the distortion of the reflected image is 4% or less, multiple spots can be measured simultaneously. If the distortion is 3% or less, and 2% or less, two-dimensional surface plasmon resonance measurement can be performed with high accuracy regardless of various conditions such as spot size, spot interval, and number of spots. It is. In general, the smaller the spot size on the chip and the distance between the spots, the smaller the distortion must be.

The region where the distortion of the reflected image is 4% or less is preferably at least 45 mm ² , more preferably 70 mm ² or more, and particularly preferably 100 mm ² or more.

  Further, the region is preferably rectangular or square on the chip, and the aspect ratio is preferably 0.5 or more, more preferably 0.6 or more, and particularly preferably 0.7 or more.

Further, the upper limit of the area of the region is not particularly defined, but is 10,000 mm ² (100 mm × 100 mm), and further 2500 mm ² (50 mm × 50 mm).

In the present invention, the absolute values of the vertical distortion and the horizontal distortion are both 0.5% or less. These distortions are calculated from the parameters shown in FIG.
Longitudinal distortion (%)
= ((H _m- (H _l + H _r ) / 2) / H _m ) × 100 Equation (2)
H _l: from the top spot upper end of the leftmost column to the lowest spot bottom length H _r: distance from the top spot upper end of the rightmost column to the lowest spot lower H _m: bottom from the top spot upper end of the central column Length to the bottom of the spot (if there is no center column, select the two left and right columns closest to the center and use the average as the center column)
Lateral distortion (%)
= ((L _m- (L _u + L _b ) / 2) / L _m ) × 100 Equation (3)
L _u : Length from the left end of the leftmost spot in the top row to the right end of the rightmost spot L _b : Length from the left end of the leftmost spot in the bottom row to the right end of the rightmost spot L _m : Left end of the leftmost spot in the center row To the right edge of the rightmost spot (if there is no center step, select the two upper and lower steps closest to the center and use the average as the center step)
When the distortion is larger than 0.5%, even if the distortion is small, a part of the spot is displaced from the mask, and accurate measurement cannot be performed, or it is not preferable because a manual operation for masking is required.

  Conventionally, when measuring an array with a two-dimensional SPR, it was only possible to process several samples at the same time, so even if distortion or distortion is outside the scope of the present invention, there is no problem in the measurement and it simply looks good. It was a recognition that it is better to have no distortion. On the other hand, when performing simultaneous measurement by two-dimensional SPR in a multi-sample (multi-spot) system as in the present invention, it is critical that numerical values such as distortion and distortion are within a predetermined range.

  Examples of means for realizing the present invention include a case where a lens having a long focal length is used as a camera lens and imaging is performed from a distance, a case where a tilt lens is used, and the like. In view of stability, lens compactness, image brightness, depth of field, and the like, a method using a telecentric lens as the camera lens is most preferable. The term “telecentric lens” as used herein refers to a lens in which the principal ray is parallel to the optical axis by providing a stop at the focal position, and among these, has object-side telecentric performance. Some lenses have image side telecentric performance, but the effect is irrelevant in the present invention. Further, even a CCTV lens which is a normal camera lens corresponds to this having object-side telecentric performance.

  If the working distance (WD) of the telecentric lens is long, a high-quality image can be obtained with a small distortion rate of the image and a large depth of field, but there is a demerit that the size of the device is large. Therefore, WD is not particularly limited, but a range of 50 to 200 mm is preferable.

  The depth of field is particularly preferably larger than the distance difference due to the inclination of the metal thin film surface. However, since there is a disadvantage that WD becomes large, it is preferable that the depth of field is at least half of the distance difference.

  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 device according to the present invention is a very high-performance device that has a low image distortion of 4% or less, an absolute value of distortion of 0.5% or less, and a clear image.

  The present invention has clarified the range required for distortion and distortion under the conditions of a spot size of 0.5 mm and a distance between spots of 0.5 mm. This also makes it possible to analyze an array having a smaller spot size and spot-to-spot distance.

  Due to the effects of the present invention, a two-dimensional surface plasmon resonance device with reduced image distortion and high performance was realized. In the future, it is expected to be widely used for screening biomolecule interactions.

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 halogen lamp (manufactured by Hosobu Inc.) was used as the light source. 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 Koki Co., Ltd.) having a diameter of 200 μm is placed at that position, and the light beam is passed through the pin hole. 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, manufactured by Sigma Koki Co., Ltd.) having an effective diameter of 24 mm. This polarized parallel light beam can irradiate a wide area.

3 nm chromium and 45 nm gold were vapor-deposited on a Lak10 slide (n _D = 1.720, manufactured by Matsunami Glass Industrial Co., Ltd.) 18 mm square and 2 mm thick. A solution prepared by dissolving 4 armPEG thiol (SUNBRIGHT PTE-100SH, manufactured by NOF Corporation) in a concentration of 1 mM in a mixed solution of 7 ml of ethanol: water = 6: 1 at a terminal functional group of a thiol group was prepared. It was soaked for 4 hours to bind 4armPEG thiol to the entire gold substrate.

  The molecular weight of 4armPEG is 10,000, and all of the four ends of PEG are thiol groups, indicating a metal bondability to gold.

  A photomask shown in FIG. 1 was placed on the slide, and irradiated with a 500 W ultra-high pressure mercury lamp (manufactured by USHIO) for 2 hours to remove 4 armPEG thiol in the UV irradiation section. The photomask has a chrome pattern on a transparent crystal with 100 chrome through-holes with a diameter of 0.5 mm. UV light is transmitted through the hole and irradiated on the slide. .

  Next, a 1 mM ethanol solution of 7-carboxyheptanethiol (7-CHT: Dojindo Laboratories) is prepared, the gold vapor deposition slide is immersed, and 7-CHT is adsorbed on the UV irradiation part to obtain a patterned chip. It was. This patterned chip was used as a test chip 1 below.

Matching oil (Cargill M series, n _D = 1.720) is attached to the glass surface of the test chip 1 and brought into contact with a 60 ° dispersion prism (Oriel 46106) with a side of 25 mm. A polarized parallel light beam was introduced. Water was brought into contact with the gold deposition 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.), and telecentric CCTV lens (MTE-55 manufactured by Moritex Corporation) After taking out only parallel light, it was photographed with a black and white CCD camera (CS8330B manufactured by Tokyo Denki Kogyo Co., Ltd.). The outline of the apparatus is shown in FIG.

The observed two-dimensional surface plasmon resonance image is shown in FIG. Since the camera is rotated 90 °, the image is also rotated 90 °, and the image corresponds to the far side of the gold thin film surface. Therefore, by comparing the spot spacing at the top of the screen with the spot spacing at the bottom of the screen, the distortion of the image due to the perspective effect can be calculated, and the value is 0.00%. Was 4% or less.
Here, the following calculation formula was used for strain calculation. The measurement points for each parameter are shown in FIG.
Strain (%) = (| L _b −L _u | / L _b ) × 100 Formula (1)
L _u : Length from the left end of the leftmost spot to the right end of the rightmost spot on the top row L _b : Length from the left end of the leftmost spot to the right end of the rightmost spot on the bottom row Further, distortion (distortion) due to lens aberration is calculated Therefore, in the screen vertical direction and the screen horizontal direction, when comparing the average of the interval between the end portions and the interval of the central portion, the vertical portion is -0.12%, the horizontal direction is + 0.25%, and the central portion The deformation was within ± 0.5%, and an image with almost no distortion was obtained. Here, the following calculation formula was used for the calculation of strain.
Longitudinal distortion (%)
= ((H _m- (H _l + H _r ) / 2) / H _m ) × 100 Equation (2)
H _l: from the top spot upper end of the leftmost column to the lowest spot bottom length H _r: distance from the top spot upper end of the rightmost column to the lowest spot lower H _m: bottom from the top spot upper end of the central column Length to the bottom of the spot (if there is no center column, select the two left and right columns closest to the center and use the average as the center column)
Lateral distortion (%)
= ((L _m- (L _u + L _b ) / 2) / L _m ) × 100 Equation (3)
L _u : Length from the left end of the leftmost spot in the top row to the right end of the rightmost spot L _b : Length from the left end of the leftmost spot in the bottom row to the right end of the rightmost spot L _m : Left end of the leftmost spot in the center row To the right edge of the rightmost spot (if there is no center step, select the two upper and lower steps closest to the center and use the average as the center step)
FIG. 5 shows an image set with a mask so that the upper left spot, the lower left spot, and the upper right spot of the obtained image are designated and the vertical and horizontal portions are divided into nine equal parts. . As a result, even with a 10 × 10 100-point spot, the masks for all 100 points could be specified without positional deviation by specifying the position of only 3 points. When these are combined, a two-dimensional surface plasmon resonance apparatus can be realized in which image distortion hardly occurs and therefore mask designation is very simple.
[Comparative example]
As an example of the conventional method, the light projecting means and the liquid contact means have the same configuration as in the embodiment, and the camera lens of the light receiving means is a lens having a single lens configuration of a spherical aromatic convex lens (doublet convex lens) instead of a telecentric lens. The result when using is shown.

  Similar to Example 1, a two-dimensional surface plasmon resonance image obtained by observing the test chip 1 is shown in FIG. Similarly, by comparing the spot spacing at the top of the screen with the spot spacing at the bottom of the screen, the distortion of the image due to perspective effects can be calculated, and the value is 4.8%, which is large on the screen, that is, on the trapezoid where the far side is small It can be seen that distortion occurs.

  Furthermore, in order to calculate distortion (distortion) due to lens aberration in the same manner as in Example 1, when comparing the average of the edge interval and the interval of the center part in each of the screen vertical direction and the screen horizontal direction, the vertical direction is 1.42%, the horizontal direction is 0.76%, which is ± 0.5% or more, and it can be seen that it is greatly distorted. Here, when calculating the distortion in the horizontal direction, the length B (the length from the left end of the leftmost spot to the right end of the rightmost spot in the center stage) is +2.4 once considering the distortion of 4.8% due to the perspective effect. % And length A (length from the left end of the leftmost spot to the right end of the rightmost spot in the uppermost row) were calculated after correction of + 4.8%.

  In addition, the three images of the upper left spot, the lower left spot, and the lower right spot are designated in the obtained image, and the image set as a mask so as to be divided into nine equal parts in the vertical and horizontal directions is shown in FIG. Shown in As a result, it can be seen that the simple three-point spot setting at equal intervals greatly shifts the mask and cannot be used practically.

100-hole photomask used in examples and comparative examples Overview of 2D surface plasmon resonance device Measurement location when calculating distortion and distortion Observation image 1 in the embodiment Equally spaced mask processing in the embodiment Observation image 1 in the comparative example Equal interval mask processing in comparative example

Claims (6)

A measuring apparatus using surface plasmon resonance, having a spot for measuring a sample on a chip and having a light receiving means for measuring reflected light from a metal thin film surface on the chip in two dimensions. A telecentric lens is used, and the working distance (WD) of the telecentric lens is in the range of 50 to 200 mm, and the region where the distortion of the reflected image is 4% or less is continuously at least 25 mm ^{2 to} 10,000 mm on the chip. A two-dimensional surface plasmon resonance apparatus having ^two or less.   2. An apparatus according to claim 1, wherein the absolute values of the longitudinal distortion and the lateral distortion are both 0.5% or less.   The apparatus according to claim 1 or 2, wherein the number of simultaneously measurable samples on the chip is 25 or more.   The apparatus according to claim 3, wherein the number of simultaneously measurable samples on the chip is 25 or more and 27889 or less.   A measurement method comprising the step of simultaneously measuring 25 or more spots on a chip using the two-dimensional surface plasmon resonance apparatus according to claim 1.   A measurement method comprising the step of simultaneously measuring spots of 25 points or more and 27889 points or less on a chip using the two-dimensional surface plasmon resonance apparatus according to claim 1 or 2.

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