JP2007256103A - Film thickness measuring method for fixing film for measuring chip, and measurement chip therefor - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a film thickness measuring method for a fixing film for a measuring chip, capable of inspecting film thicknesses of the fixing films in a large number of measurement chips, in a short time, and to provide a measuring chip therefor. <P>SOLUTION: This film thickness measuring method, for the fixing film for the measuring chip, is provided with the first region 4A region with a metal film 4 and the fixing film 6, and the second region 4B, having a reflectance serving, as a reference. In the method, a calibration chip 10 is manufactured to make the fixing film 6 have a prescribed film thickness, the film thicknesses of the fixing film 6 is measured in the calibrating chip 10 prepared in the first step; a reflected luminous energy ratio of the first region 4A to the second area 4B is found at the same time, to find a relation between the reflected luminous energy ratio and the film thicknesses of the fixing film 6, a reflected luminous energy ratio of the first region 4A to the second area 4B is found as to an inspected chip 10, having an unknown film thickness of the fixing film 6, and the film thicknesses of the fixing film 6 in the inspected chip 10 is found, based on the reflected luminous energy ratio of the calibration chip 10 found in the third step, and the reflected luminous energy ratio of the inspected chip 10 found in the fourth step. This measuring chip of the present invention is constituted to be used in the film thickness measuring method for the fixing film. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention relates to a measuring chip used in a surface plasmon resonance (SPR) measuring apparatus, a method for measuring the thickness of an immobilized film of a measuring chip for measuring the thickness of an immobilized film for immobilizing a ligand on the surface, and the immobilization. The present invention relates to a measuring chip including means for realizing a film thickness measuring method.

  For example, a measurement device using SPR is known as a measurement device for measuring the reaction of a sample when investigating the interaction of biochemical substances such as proteins and DNA, or performing drug screening.

  Surface plasmon refers to the quantum of free-electron density waves generated by the collective oscillation of free electrons in a metal and traveling along the surface of the metal.

  The SPR measurement device uses a metal film as a thin film formed on a transparent dielectric, and uses one surface of the metal film as a sensor surface, generates SPR on the sensor surface, and detects this. The reaction state of the substance generated on the sensor surface is measured.

  When light is incident on the back side of the sensor surface of the metal film so as to satisfy the total reflection condition, total reflection occurs on the light incident surface, but only a small part of the incident light passes through the metal film without being reflected. And ooze out to the sensor surface. The light wave that oozes out on the sensor surface is called an evanescent wave. When the evanescent wave and the surface plasmon frequency coincide with each other and resonate, the intensity of the reflected light is greatly reduced. The SPR measurement device detects the SPR generated on the sensor surface on the back side by capturing the attenuation of the reflected light reflected by the light incident surface.

  The incident angle or resonance angle of light for generating SPR depends on the refractive index of the medium through which the evanescent wave and the surface plasmon propagate. In other words, if the refractive index of the medium changes, the resonance angle that generates SPR also changes. The substance in contact with the sensor surface becomes a medium for propagating evanescent waves and surface plasmons. For example, when a chemical reaction such as bonding or detachment between two types of molecules occurs on the sensor surface, it is regarded as a change in the refractive index of the medium. Appears and the resonance angle changes. The SPR measurement device measures the interaction between molecules by capturing the change in the resonance angle.

  In experiments and research in the biochemical field, proteins, DNA, drugs, etc. are used as ligands and analytes. For example, when screening for drugs, biological substances such as proteins are used as ligands, which are fixed to the sensor surface, and the sensor surface is contacted with multiple types of drugs that are analytes, and their interaction is determined. Investigate.

  The measurement chip is generally a transparent dielectric block such as glass or plastic, a metal film formed on the surface of the dielectric block, and an immobilization film formed thereon and having a functional group capable of immobilizing a ligand. Consists of. In the measurement chip, the ligand is fixed to the metal film through the functional group of the fixed film.

  Some SPR measurement apparatuses employ a Kreschmann arrangement as an optical system for making light incident on a metal film (Patent Document 1). The SPR measurement device has a light source that generates a light beam and the light beam generated by the light source at various angles to the dielectric block so as to totally reflect the light beam at the interface between the dielectric block and the metal film of the measurement chip. An optical system for incidence is provided, and light detection means for detecting the SPR state, that is, the state of total reflection attenuation, by measuring the intensity of the light beam totally reflected at the interface.

  In order to obtain various incident angles in the SPR measurement device, for example, a relatively thick light beam may be incident on the interface in a converged state so that components incident on the light beam at various angles are included. . In this case, it can be detected by an area sensor extending in a direction in which all light beams reflected at various reflection angles can be received (for example, Patent Document 2).

  Further, there are cases where it is desired to obtain two-dimensional physical property information of a sample. However, application of the SPR measurement device to such a use is being studied.

For example, taking a two-dimensional physical property analysis of a sample by an SPR measuring device as an example, when a parallel light flux having a predetermined wavelength is incident on a region having a two-dimensional spread of the interface, Among these, a region in which the refractive index of the sample is a refractive index that causes total reflection attenuation at the incident angle and the wavelength is detected as dark transition. Therefore, using a parallel light beam having a plurality of wavelengths having a somewhat wide light beam cross section, a desired single wavelength parallel light beam is selected from the parallel light beams, and is incident on the interface between the dielectric block of the measurement chip and the metal film, By detecting the light intensity distribution in the cross section of the parallel light beam totally reflected at the interface, it is possible to measure characteristics related to the two-dimensional refractive index distribution of the sample in the plane along the interface (Patent Documents 3 to 5). .
JP-A-6-167443 JP 11-326194 A JP-T-2004-531719 JP-T-2005-515424 JP 2004-117298 A

  However, as described above, the SPR measurement technique measures a change in the refractive index in the vicinity of the metal film of the measurement chip. Therefore, not only the binding amount of the analyte but also the film thickness of the immobilized film that fixes the ligand. The measured value is different.

  Therefore, even if the binding amount of the analyte to be measured is the same, if the thickness of the immobilized film is different, the refractive index in the vicinity will also be different. There is a problem that the value cannot be obtained.

  The present invention has been made to solve the above problems, and in the measurement chip used for quantifying the analyte using SPR, the thickness of the immobilized film of the measurement chip to be manufactured is In order to inspect the film thickness of a large number of measurement chips in a simple way to determine whether they are within a specified error range and to exclude non-standard measurement chips and to provide only reliable measurement chips An object of the present invention is to provide a method for measuring the thickness of an immobilized film of a measurement chip that can be used, and a measurement chip including means for realizing the method for measuring the thickness of an immobilized film.

  The invention according to claim 1 is formed in at least a partial region of the surface of the substrate, and has a metal film having an optimum thickness for causing surface plasmon resonance by light having a predetermined wavelength and incident angle, A first region in which a fixed film for fixing a ligand that interacts with a predetermined analyte is formed on the surface of the metal film; and provided adjacent to the first region, for light of the wavelength and incident angle A measurement chip including a second region having a reference reflectivity, the first step of producing a calibration chip in which the immobilization film in the first region has a predetermined film thickness, and the first step. A second step of measuring the thickness of the immobilization film in the first region of the calibration chip, and simultaneously measuring the amount of reflected light in the first region and the second region for the calibration chip; Said A third step of obtaining a reflected light amount ratio, which is a ratio of a reflected light amount to one region, and then obtaining a relationship between the reflected light amount ratio and the film thickness of the immobilizing film; For the chip to be inspected whose film thickness is unknown, the reflected light amount in the first region and the second region is simultaneously measured, and the reflected light amount ratio of the first region to the second region is obtained in the fourth step and the third step. And a fifth step of determining the film thickness of the chip to be inspected based on the ratio of the reflected light quantity of the calibration chip determined and the ratio of the amount of reflected light of the chip to be inspected determined in the fourth step. The present invention relates to a method for measuring the thickness of a fixed film of a chip.

  In the method for measuring the thickness of the fixed film, the thickness of the fixed film is measured for a calibration chip in which the fixed film in the first region has a predetermined thickness among the measurement chips, and The reflected light amount ratio between the first region and the second region is measured, and the relationship between the film thickness of the fixed film and the reflected light amount ratio is obtained.

  Next, the reflected light amount ratio between the first region and the second region is measured for a chip to be inspected whose thickness of the immobilization film in the first region is unknown among the measurement chips.

  Finally, from the relationship between the reflected light amount ratio measured at the chip to be inspected and the film thickness of the fixing film and the reflected light amount ratio obtained for the calibration chip, immobilization in the first region of the inspection chip is performed. Obtain the film thickness.

  As described above, only the reflected light amount ratio is actually measured for the chip to be inspected, and since the film thickness of the immobilization film is not directly measured, a large number of chips to be inspected can be processed in a short time.

  Therefore, for a large number of measurement chips, since the film thickness of the fixed film can be separated into those having a predetermined error range and those whose film thickness is not specified in a short time, only a highly reliable measurement chip can be provided. .

  In addition, since the method for measuring the film thickness of the immobilization film obtains the film thickness of the chip to be inspected based on the reflected light amount ratio in this way, the measurement result is not influenced by the amount of incident light. Therefore, the measurement results obtained are highly reliable.

  According to a second aspect of the present invention, in the first step, the base is coated with a metal film having an optimum thickness for generating surface plasmon resonance when light having the predetermined wavelength and incident angle is incident. A first region and a second region formed by coating the base with a metal film that is not coated with a metal film or having a thickness different from that of the metal film formed in the first region; The method according to claim 1, wherein the first and second regions are formed with a calibration chip in which the first and second regions are covered with a fixed film having a uniform thickness.

  In the calibration chip manufactured in the first step of the film thickness measurement method, the first region is optimized so that SPR occurs with respect to light having a predetermined wavelength and incident angle. Such optimization is not performed for the two areas.

  Therefore, when the calibration chip is irradiated with the light having the wavelength and the incident angle, the SPR is generated in the first region and the reflected light amount is remarkably reduced, whereas the SPR is hardly generated in the second region. Does not decrease as significantly as the first region.

  In the calibration chip, not only when the ligand immobilized on the immobilized film interacts with alanite, but also when the thickness of the immobilized film changes, the incident angle, that is, the resonance angle at which SPR occurs is increased or decreased. To do. If the wavelength and the incident angle of the incident light are fixed, the increase or decrease in the resonance angle caused by the change in the thickness of the immobilized film is observed as an increase or decrease in the amount of reflected light.

  Therefore, in the calibration chip, when the film thickness of the immobilization film changes in the first region, the amount of reflected light increases or decreases significantly, whereas in the second region, the film thickness of the immobilization film changes. However, since the amount of reflected light hardly changes, the ratio of the amount of reflected light between the first region and the second region increases or decreases significantly when the thickness of the immobilization film changes.

  Therefore, a plurality of calibration chips having different thicknesses of the fixed film are prepared, the reflected light amount ratio between the first region and the second region is obtained for each calibration chip, and the reflected light amount ratio is determined as the fixed film thickness. By plotting against the thickness, the relationship between the thickness of the immobilization film and the reflected light amount ratio can be obtained.

  In the calibration chip used in the method for measuring the thickness of the immobilization film, the thickness is smaller than the optimum thickness for generating SPR for the light having the predetermined wavelength and incident angle on the entire surface of the substrate. A metal film is formed as a second region, and in a part of the substrate, a metal film is further formed on the metal film to generate an SPR for light having a predetermined wavelength and incident angle. The first region can be formed by setting the thickness to be optimal. Further, only a partial region of the substrate is described as a first region by covering with a metal film having an optimum thickness for generating surface plasmon resonance for light of a predetermined wavelength and incident angle, and the remaining region is It is good also as a 2nd field.

  Next, the substrate in which the first region and the second region are formed is immersed in the order of the SAM solution, the epoxidizing agent, the dextran solution, and the bromoacetic acid solution to fix the first region and the second region to a uniform thickness. A calibration chip can be produced by coating with a chemical film.

  As described above, the calibration chip can be easily manufactured because it is sufficient to cover the whole with the fixed film having a uniform thickness.

  According to a third aspect of the present invention, in the first step, the substrate is coated with a metal film having an optimum thickness for generating surface plasmon resonance with respect to light having the predetermined wavelength and incident angle. 2. A calibration chip having a first region in which a surface of the metal film is covered with an immobilization film and a second region in which the surface of the metal film is not covered with an immobilization film is produced. The present invention relates to a method for measuring a film thickness of an immobilized film of the measurement chip.

  In the calibration chip produced in the first step of the method for measuring the thickness of the immobilized film, the metal film is covered with the immobilized film in the first region, and the metal film is exposed in the second region. In the second region, SPR is generated for light incident at the resonance angle, and the amount of reflected light is reduced. In contrast, in the first region, although SPR occurs with respect to the light, the resonance angle is different from that of the second region. Therefore, if the wavelength and incident angle of incident light are fixed at the wavelength and incident angle at which SPR occurs in the second region, the change in the resonance angle accompanying the change in the thickness of the immobilized film can be regarded as an increase or decrease in the amount of reflected light.

  Therefore, also in the calibration chip, a plurality of first regions having different thicknesses of the fixing film are formed, or a plurality of calibration chips having different thicknesses of the fixing film formed in the first region are formed. Then, if the reflected light amount ratio in the first region and the second region is obtained and plotted against the film thickness of the immobilized film, the relationship between the film thickness of the immobilized film and the reflected light amount ratio is obtained.

  In the method for measuring the thickness of the immobilized film, the entire surface of the substrate is covered with a metal film having a thickness that causes SPR at a predetermined resonance angle, and a part of the metal film is covered with the immobilized film. Since the calibration chip is manufactured by covering the first region, the calibration chip is calibrated with fewer steps than the calibration chip used in the method for measuring the thickness of the immobilized film according to claim 2. A chip can be produced.

  According to a fourth aspect of the present invention, in the fourth step, a two-dimensional distribution of the reflected light amount ratio between the second region and the first region is obtained for the chip to be inspected, and in the fifth step, the third step The two-dimensional distribution of the film thickness in the first region of the chip to be inspected is calculated based on the ratio of the reflected light quantity of the calibration chip obtained in step 4 and the two-dimensional distribution of the reflected light quantity ratio of the chip to be inspected obtained in the fourth step. It is related with the film thickness measuring method of the fixed film of the measuring chip according to any one of claims 1 to 3.

  In the immobilization film thickness measurement method, since the two-dimensional distribution of the immobilization film thickness in the first region of the chip to be inspected is obtained, the immobilization film is inspected for the immobilization film thickness measurement of the measurement chip. By applying the film thickness measurement method, it is possible to effectively eliminate a measurement chip having a non-uniform thickness distribution of the fixed film.

  According to a fifth aspect of the present invention, there is provided a metal film that is formed in at least a partial region of the surface of a substrate and has a thickness that is optimal for causing surface plasmon resonance by light having a predetermined wavelength and an incident angle; A first region in which a fixed film for fixing a ligand that interacts with a predetermined analyte is formed on the surface of the metal film; and provided adjacent to the first region, for light of the wavelength and incident angle The present invention relates to a measurement chip including a second region having a reference reflectance.

  In the measurement chip, the amount of reflected light increases or decreases in the first region if the thickness of the immobilized film changes, whereas the amount of reflected light in the second region does not change even if the thickness of the immobilized film changes. Constant. Therefore, the reflected light amount ratio between the first region and the second region also increases or decreases as the thickness of the immobilization film in the first region changes.

  Here, the reflected light amount ratio does not depend on the amount of incident light.

  Therefore, if a calibration chip in which the film thickness of the immobilization film in the first region is changed and the film thickness measurement method for the immobilization film according to claim 1 is performed, the amount of incident light depends on the amount of incident light. Therefore, the film thickness of the measuring chip can be measured.

  Further, if the immobilization film and the analyte interact, the amount of reflected light changes in the first region, but the amount of reflected light is constant in the second region. By obtaining the reflected light amount ratio, the analyte can be detected without being affected by the amount of incident light.

  According to a sixth aspect of the present invention, in the first region, the substrate is coated with a metal film having an optimum thickness for causing surface plasmon resonance when light having the predetermined wavelength and incident angle is incident. In the second region, the substrate is not covered with a metal film, or is coated with a metal film having a thickness different from that of the metal film formed in the first region. The measurement chip according to claim 5, wherein the second region is covered with a fixed film having a uniform thickness.

  As described in the second aspect, in the measurement chip, SPR occurs in the first region with respect to the light having the wavelength and the incident angle, but no SPR occurs in the second region. In the first region, if the film thickness of the immobilization film changes, the resonance angle at which SPR occurs also changes. As a result, the amount of reflected light increases or decreases. In the second region, the film thickness of the immobilization film. Regardless of the case, the amount of reflected light is constant.

  Therefore, also in the measurement chip, the ratio of the amount of reflected light between the first region and the second region changes corresponding to the thickness of the immobilized film, and therefore the thickness of the immobilized film is known in the measurement chip. Is suitably used as a calibration chip in the method for measuring the thickness of an immobilized film according to claims 1 to 4.

  According to a seventh aspect of the present invention, a substrate is coated with a metal film having an optimum thickness for causing surface plasmon resonance when light having the predetermined wavelength and incident angle is incident, and the first The measurement chip according to claim 5, wherein an immobilization film is formed on a surface of the metal film in the region, and a surface of the metal film is not covered with the immobilization film in the second region.

  In the measurement chip, in the second region, SPR occurs with respect to the light having the wavelength and the incident angle, and the amount of reflected light is significantly reduced. However, since no immobilization film is formed in the second region, the amount of reflected light is not affected by the film thickness of the immobilization film, and is constant if the wavelength, incident angle, and amount of incident light are constant. Can be obtained.

  Therefore, also in the measurement chip, the ratio of the amount of reflected light between the first region and the second region changes corresponding to the thickness of the immobilized film, and therefore the thickness of the immobilized film is known in the measurement chip. Is suitably used as a calibration chip in the method for measuring the thickness of an immobilized film according to claims 1 to 4.

  As described above, according to the present invention, the method for measuring the thickness of the fixed film of the measurement chip that can easily measure and inspect the thickness of the fixed film of the measurement chip, and the measurement chip having means for realizing the measurement method Is provided.

1. Embodiment 1
As shown in FIG. 1, the measuring chip 10 according to the first embodiment includes a long plate-like base 2, a metal film 4 formed on one surface of the base 2, and a fixed formed on the surface of the metal film 4. And a chemical film 6.

  The substrate 2 is a dielectric that is transparent to incident light having a wavelength of 532 nm, which is an example of the light of the present invention, such as optical glass such as quartz glass and borosilicate glass, and optical resin such as polycarbonate resin and cyclic polyolefin resin. Can be formed from And as shown in FIG. 2, the cross section cut | disconnected by plane XX 'along the width direction goes to the surface where the metal film 4 is not formed from the surface where the metal film 4 is formed. The prism shape is reduced.

  The metal film 4 is not particularly limited as long as it can cause SPR. For example, the metal film 4 is formed by sputtering a metal selected from gold, silver, copper, and aluminum on the wide surface of the base 2. Gold and silver are preferable from the viewpoint of difficulty of plasmon attenuation, and gold is particularly preferable from the viewpoint of excellent stability against oxidation.

  The metal film 4 has a first region 4A formed to a thickness that causes surface plasmon resonance when incident light having a wavelength of 532 nm is incident at a predetermined incident angle, for example, an incident angle of 54 degrees with respect to the reflecting surface; The second region 4B is thinner than the first region 4A. The thickness t1 of the first region 4A in the metal film 4 is, for example, 45 nm, and the thickness t2 of the second region 4B is, for example, 15 nm.

  The measuring chip 10 can be manufactured according to the following procedure.

  First, as shown in FIG. 3, the sample base 200 of the sputter deposition apparatus (not shown) has 24 bases 2 so that the wide side of the base 2 faces the sputter target (not shown). Place on top. The 24 substrates 2 are divided into four groups, and each group is arranged at equal intervals. In each group, six substrates 2 are arranged in close contact with each other in the width direction.

  When the substrate 2 is arranged on the sample stage 200 of the sputter deposition apparatus, the mask 20 is placed in close contact with the substrate 2. As shown in FIG. 4, the mask 20 has four continuous slit regions 21 in which six continuous slits 22 are formed at regular intervals along a substantially radial direction. The continuous slit 22 is formed so as to be continuous over the entire length of the base 2. And the intermittent slit area | region 23 in which the six intermittent slits 24 were formed at equal intervals along the substantially radial direction just in the middle of the two adjacent continuous slit area | regions 21 is formed. Therefore, four intermittent slit regions 23 are also formed at regular intervals in the same manner as the continuous slit region 21. Unlike the continuous slit 22, the intermittent slit 24 is formed to be intermittent in a broken line shape.

  First, as shown in FIG. 5A, the mask 20 is arranged so that the intermittent slit region 23 is located above the base 2. As a result, the intermittent slit 24 is positioned above each base body 2. By sputtering gold to a thickness of 30 nm in this state, the first region 4A is intermittently formed on the wide surface of the substrate 2 as shown in FIG.

  When the first region 4A is formed, the mask 20 is rotated 45 degrees from the state of FIG. 5, and the continuous slit region 21 is disposed above the base 2 as shown in FIG. . As a result, the continuous slits 22 are positioned above the respective substrates 2. By sputtering gold to a thickness of 15 nm in this state, the first region 4A and the second region 4B are formed on the wide surface of the substrate 2 as shown in FIG. 6B.

  When the metal film 4 is formed on the substrate 2, the measurement chip 10 is manufactured by forming the immobilization film 6 on the surface of the metal film 4 according to the procedure shown in FIG. Below, the procedure which produced the measurement chip | tip 10 is demonstrated.

  A 5.0 mM solution of 11-hydroxy-1-undecanthiol in ethanol / water (80/20) was added so as to contact the metal film 4 and surface treatment was performed at 25 ° C. for 18 hours. Then, it was washed 5 times with ethanol, once with a mixed solvent of ethanol / water, and 5 times with water.

  Next, the surface coated with 11-hydroxy-1-undecanethiol was brought into contact with a 10 wt% epichlorohydrin solution (solvent: 1: 1 mixed solution of 0.4 M sodium hydroxide and diethylene glycol dimethyl ether), and 25 The reaction was allowed to proceed for 4 hours in a shaking incubator at 0 ° C. Thereafter, the surface was washed twice with ethanol and five times with water.

  Next, 4.5 ml of sodium hydroxide was added to 40.5 ml of 25% by weight dextran (T500, manufactured by Pharmacia), and the solution was brought into contact with the epichlorohydrin-treated surface. It was then incubated for 20 hours at 25 ° C. in a shaking incubator. And the surface was washed 10 times with 50 degree water.

  Subsequently, a mixture of 3.5 g of bromoacetic acid dissolved in 27 g of 2M sodium hydroxide solution was brought into contact with the dextran-treated surface and incubated at 28 ° C. for 16 hours in a shaking incubator. After incubation, the surface was washed with water and the above procedure was repeated once.

  One of the measurement chips 10 thus produced is used as the calibration chip 10 and the thickness of the immobilization film is measured with an ellipsometer.

  The calibration chip 10 whose film thickness was measured with an ellipsometer was attached to the measuring apparatus 100, the two-dimensional distribution of the reflected light amount was measured, and the relationship between the film thickness and the reflected light amount was determined.

  As shown in FIG. 8, the measuring apparatus 100 includes a measuring chip holder 102 on which the calibration chip 10 is mounted, and a light source 104 that irradiates parallel light toward the calibration chip 10 mounted on the measuring chip holder 102. The CCD camera 106 that measures the intensity of the reflected light from the calibration chip 10 and the telecentric lens 108 that converges the reflected light from the calibration chip 10 on the CCD camera 106 are provided. A polarizing filter 110 that transmits only p-polarized light is provided on the exit surface of the light source 104, and an interference filter 112 that transmits only light having a wavelength of 532 nm is provided on the incident surface of the telecentric lens 108. Further, a mask 114 that restricts the width of light emitted from the light source 104 is inserted between the emission surface of the light source 104 and the polarizing filter 110.

  The calibration chip 10 is measured so that the surface on the side of the base 2 having a small width, in other words, the surface opposite to the side on which the metal film 4 and the fixing film 6 are formed faces the light source 104 and the CCD camera 106. Attached to the chip holder 102.

  Hereinafter, the operation of the measuring apparatus 100 will be described. As shown in FIG. 8, the light emitted from the light source 104 enters the metal film 4 from the surface of the calibration chip 10 that does not have the metal film 4 at an incident angle θ and is reflected by the metal film 4. Then, the light enters the telecentric lens 108 and the CCD camera 106 at the same emission angle. The base 2 of the measurement chip 10 functions as a prism, and guides the light incident on the base 2 to the side of the metal film 4 in contact with the base 2 at an incident angle θ.

  As shown in FIG. 9, when the incident angle θ of light with respect to the metal film 4 is a specific value, for example, about 48 to 52 degrees, the first region 4A of the metal film 4, that is, the region having a thickness of 45 nm is incident. SPR occurs due to light, and the amount of reflected light is greatly attenuated. The incident angle θ at which the amount of reflected light has the lowest value is 48 degrees when the fixing film 6 is not formed on the calibration chip 10 as shown by the thick solid line in the figure, and the fixing film 6 having a thickness of 5 nm is formed. In this case, it is 50 degrees as shown by a thick broken line in the figure, and when the fixed film 6 having a thickness of 10 nm is formed, it is 52 degrees as shown by a thick dashed line in the figure.

  When the incident angle θ is larger than the above angle, the reflectance increases regardless of the thickness of the immobilization film 6, but the immobilization film 6 is not formed and the immobilization film 6 has a thickness of 5 nm. The difference in reflectance from the case where the thickness of the immobilizing film 6 is 10 nm is maximized when the incident angle θ is around 54 degrees.

  On the other hand, in the second region 4B where the thickness of the metal film is 15 nm, when the incident angle θ is 54, the reflectance is about 0. As shown by the thin solid line, the broken line, and the alternate long and short dash line in FIG. 4 and the thickness of the immobilizing film 6 are almost the same.

  The relationship between the reflectance when the incident angle θ is 54 degrees and the film thickness of the immobilization film 6 is shown for the first region 4A having a thickness of 45 nm and the second region 4B having a thickness of 15 nm in the metal film 4. 10 (A). In FIG. 10A, the first region 4A is indicated by a broken line, and the second region 4B is indicated by a solid line. As can be seen from FIG. 10A, the reflectance of the first region 4A is about 0.205 when the thickness of the immobilizing film 6 is 0 nm, and is about 0.14 when the thickness is 5 nm. In this case, it is about 0.08, and decreases linearly as the thickness of the immobilizing film 6 increases. On the other hand, in the second region 4B, the reflectance is substantially constant at 0.41 regardless of the thickness of the immobilization film 6. In actual measurement, the reflectance cannot be obtained directly from the reflected light distribution of one two-dimensional image. Therefore, a reflected light amount ratio, which is a ratio of reflectance between the first region 4A and the second region 4B, was obtained. The reflected light amount ratio indicates that the lower the value, the stronger the contrast between the first region 4A and the second region 4B. The relationship between the reflected light amount ratio and the thickness of the immobilization film 6 is shown in FIG. As shown in FIG. 10B, the reflected light amount ratio is about 0.5 when the thickness of the immobilizing film 6 is 0 nm, about 0.35 when it is 5 nm, and about 0.35 when it is 10 nm. As the thickness of the immobilizing film 6 increased to 0.2, it decreased linearly. Thus, it can be seen that the decrease in the reflected light amount ratio when the thickness of the immobilization film 6 is increased is more significant than the decrease in the reflectance.

  Next, the reflectance of the metal film 4 is measured with the measuring device shown in FIG. 8 with respect to the measuring chip 10 whose thickness of the immobilizing film 6 shown in FIG. 11 is unknown, and the amount of reflected light between the first region 4A and the second region 4B. The ratio was determined. Note that the cross section of the measurement chip of FIG. 11 taken along the cross section X-X ′ in the width direction is as shown in FIG. 2, and four first regions 4 </ b> A are provided. And the fixed film | membrane 6 is 4 area | regions of the film thickness evaluation line 1, the film thickness evaluation line 2, the film thickness evaluation line 3, and the film thickness evaluation line 4 corresponding to four areas | regions of the 1st area | region 4A by the 2nd area | region 4B. It is divided into. In FIG. 11, the high color density portions of the film thickness evaluation line 1, the film thickness evaluation line 2, the film thickness evaluation line 3, and the film thickness evaluation line 4 indicate that the film thickness is thick, and the low color density portion indicates the film. Indicates that the thickness is thin.

  FIG. 12A shows the reflectance distribution in each region of the film thickness evaluation line 1, the film thickness evaluation line 2, the film thickness evaluation line 3, and the film thickness evaluation line 4. Then, from the distribution of reflectance in each region of the measurement chip 10 shown in FIG. 12A and the relationship between the reflectance shown in FIG. The thickness distribution of the immobilization film 6 was obtained. The results are shown in FIG.

As described above, according to the method shown in the first embodiment, the thickness distribution of the immobilization film 6 can be obtained only by measuring the amount of reflected light of the metal film 4 with the measurement chip 10.
2. Embodiment 2
As shown in FIG. 13, the measuring chip 11 according to the second embodiment is formed on a long plate-like base 12, a metal film 14 formed on one surface of the base 12, and a surface of the metal film 14. And an immobilization film 16.

  The base 12 is formed of a dielectric that is transparent to the light emitted from the light source 104 of the measuring apparatus 100, similarly to the base 2 provided in the measurement chip 10 according to the first embodiment, and is on the side on which the metal film 14 is formed. It has a prism-shaped cross section that decreases from the surface toward the opposite surface. As the dielectric material used for forming the substrate 12, the same material as described in the first embodiment is used.

  Unlike the metal film 4 in the first embodiment, the metal film 14 is a uniform film of gold, silver, copper, or aluminum having a thickness of 45 nm. The material of the metal film 14 is preferably gold or silver, and most preferably gold.

  An immobilization film 16 is formed on the surface of the metal film 14, and the immobilization film 16 is divided into six regions.

  The measurement chip 11 can be manufactured according to the following procedure.

  First, as shown in FIG. 3, the sample base 200 of the sputter deposition apparatus (not shown) is configured so that the 24 bases 12 are opposed to the sputter target (not shown) on the wide side of the base 2. Place on top. The 24 substrates 12 are divided into four groups, and each group is arranged at equal intervals. In each group, six bases 12 are arranged in close contact with each other in the width direction.

  When the substrate 12 is arranged on the sample stage 200 of the sputter deposition apparatus, a mask 30 is placed in close contact with the substrate 12 as shown in FIG. As shown in FIG. 14, the mask 30 has four continuous slit regions 31 each having six continuous slits 32 formed at equal intervals along the substantially radial direction.

  Sputtering gold to a thickness of 45 nm with the mask 30 in intimate contact with the substrate 12 forms a metal film 14 on the wide surface of the substrate 12 as shown in FIG. .

  When the metal film 14 is formed, as shown in FIG. 16A, a silicone rubber seal is formed on a cuvette body in which holes having the same shape and size as the intermittent slits 24 of the mask 20 are formed in a fluorine resin sheet. The stacked materials were stacked on the region of the substrate 12 where the fixing film 16 was to be formed, and six cuvettes 18 corresponding to the regions were formed. In addition, instead of a laminate of the fluororesin sheet and silicone rubber, a hole made in a highly chemical-resistant rubber such as silicone rubber or fluororubber and the cuvette body and sealing material integrated is used. It may be used.

  Next, a 5.0 mM solution of 11-hydroxy-1-undecanthiol in ethanol / water (80/20) was injected into each cuvette 18 and surface treatment was performed at 25 ° C. for 18 hours. Then, it was washed 5 times with ethanol, once with a mixed solvent of ethanol / water, and 5 times with water.

  Then, the surface coated with 11-hydroxy-1-undecanthiol was brought into contact with a 10 wt% epichlorohydrin solution (solvent: 1: 1 mixed solution of 0.4 M sodium hydroxide and diethylene glycol dimethyl ether), and 25 ° C. The reaction was allowed to proceed for 4 hours in a shaking incubator. Thereafter, the surface was washed twice with ethanol and five times with water.

  Next, 4.5 ml of sodium hydroxide was added to 40.5 ml of 25% by weight dextran (T500, manufactured by Pharmacia), and the solution was brought into contact with the epichlorohydrin-treated surface. It was then incubated for 20 hours at 25 ° C. in a shaking incubator. And the surface was washed 10 times with 50 degree water.

  Subsequently, a mixture of 3.5 g of bromoacetic acid dissolved in 27 g of 2M sodium hydroxide solution was brought into contact with the dextran-treated surface and incubated at 28 ° C. for 16 hours in a shaking incubator. After incubation, the surface was washed with water and the above procedure was repeated once.

  In the measurement chip 11 formed in this way, the relationship between the thickness of the immobilization film 16 and the reflectance of the metal film 14 is obtained using the area in the metal film 14 where the immobilization film 16 is not formed as a reference area. Can do. The procedure for obtaining the relationship and the measuring apparatus are as described in the first embodiment.

FIG. 1 is a longitudinal sectional view showing the configuration of the measurement chip according to the first embodiment. FIG. 2 is a cross-sectional view in the width direction illustrating the configuration of the measurement chip according to the first embodiment. FIG. 3 is a process diagram illustrating a procedure for producing a metal film on a base body included in the measurement chip according to the first embodiment. FIG. 4 is a plan view illustrating a mask used for forming a metal film on a base body included in the measurement chip according to the first embodiment. FIG. 5 is a process diagram illustrating a procedure for producing a metal film on a base body included in the measurement chip according to the first embodiment. FIG. 6 is a process diagram illustrating a procedure for forming a metal film on a base body included in the measurement chip according to the first embodiment. FIG. 7 is a process diagram showing a procedure for forming an immobilization film on the surface of the metal film formed on the substrate. FIG. 8 is a schematic diagram showing a configuration of a measuring apparatus to which the measuring chip shown in FIG. 1 is attached. FIG. 9 is a graph showing the relationship between the incident angle of light and the reflectance in the thick first region and the thin second region in the metal film of the measurement chip shown in FIG. FIG. 10 is a graph showing the relationship between the thickness of the immobilized film, the reflectance, and the reflected light amount ratio in the measurement chip shown in FIG. FIG. 11 is a plan view showing an outline of a measurement chip used for obtaining the thickness of the immobilized film itself from the relationship between the reflectance of the metal film and the thickness of the immobilized film. FIG. 12 is a graph showing the distribution of the reflected light amount ratio between the first region and the second region in the measurement chip shown in FIG. 11 and the film thickness distribution obtained from the distribution of the reflected light amount ratio. FIG. 13 is a longitudinal cross-sectional view showing the configuration of the measurement chip according to the second embodiment. FIG. 14 is a plan view showing a mask used for producing a metal film on a base body included in the measurement chip according to the second embodiment. FIG. 15 is a process diagram illustrating a procedure for producing a metal film on a base body included in the measurement chip according to the second embodiment. FIG. 16 is a process diagram showing a procedure for forming an immobilization film on the surface of a metal film formed on a substrate in the measurement chip according to the second embodiment.

Explanation of symbols

2 Base 4 Metal film 4A 1st area 4B 2nd area 6 Immobilization film 10 Measurement chip 11 Measurement chip 12 Base 14 Metal film 16 Immobilization film 18 Cuvette 20 Mask 21 Continuous slit area 22 Continuous slit 23 Intermittent slit area 24 Intermittent slit DESCRIPTION OF SYMBOLS 30 Mask 31 Continuous slit area 32 Continuous slit 100 Measuring apparatus 102 Measuring chip holder 104 Light source 106 CCD camera 108 Telecentric lens 110 Polarizing filter 112 Interference filter 114 Mask 200 Sample stand

Claims (7)

A metal film formed in at least a partial region of the surface of the substrate and having an optimum thickness for causing surface plasmon resonance by light of a predetermined wavelength and incident angle; and a predetermined analyte on the surface of the metal film A first region on which an immobilization film for immobilizing a ligand that interacts with the first region is formed; and a second region that is provided adjacent to the first region and has a reflectance that serves as a reference for light having the wavelength and the incident angle. A first step of producing a calibration chip having a predetermined thickness of the immobilization film in the first region,
A second step of measuring the thickness of the immobilization film in the first region of the calibration chip produced in the first step;
For the calibration chip, the amount of reflected light in the first region and the second region is simultaneously measured to obtain a reflected light amount ratio that is a ratio of the reflected light amount between the first region and the second region, and then the reflected light amount ratio And a third step for determining the relationship between the thickness of the fixed film and the immobilized film;
With respect to the chip to be inspected whose thickness of the immobilization film is unknown, the reflected light amount in the first region and the second region is simultaneously measured, and the reflected light amount ratio between the first region and the second region is measured. A fourth step for determining
Based on the reflected light amount ratio of the calibration chip obtained in the third step and the reflected light amount ratio of the chip to be inspected obtained in the fourth step, the thickness of the immobilization film in the first region of the chip to be inspected is determined. And a method for measuring the thickness of the fixed film of the measuring chip.   In the first step, a first region in which a substrate is coated with a metal film having an optimum thickness for generating surface plasmon resonance for light of the predetermined wavelength and incident angle; and the substrate is a metal film. A second region which is not covered or is covered with a metal film having a thickness different from that of the metal film formed in the first region, and the first and second regions have a uniform thickness. The method for measuring a film thickness of an immobilized film of a measurement chip according to claim 1, wherein a calibration chip coated with a fixed film is prepared. In the first step, the substrate is coated with a metal film having an optimum thickness for causing surface plasmon resonance with respect to the light having the predetermined wavelength and incident angle,
2. The calibration chip according to claim 1, wherein the calibration chip has a first region in which a surface of the metal film is covered with an immobilization film and a second region in which the surface of the metal film is not covered with an immobilization film. A method for measuring the thickness of the fixed film of the measuring chip. In the fourth step, a two-dimensional distribution of the reflected light amount ratio between the second region and the first region is obtained for the chip to be inspected,
In the fifth step, based on the reflected light ratio of the calibration chip obtained in the third step and the two-dimensional distribution of the reflected light ratio of the chip to be inspected obtained in the fourth step, The method for measuring a film thickness of an immobilized film of a measurement chip according to any one of claims 1 to 3, wherein a two-dimensional distribution of film thickness in one region is obtained. A metal film formed in at least a partial region of the surface of the substrate and having an optimum thickness for causing surface plasmon resonance by light of a predetermined wavelength and incident angle;
A first region in which an immobilized film for fixing a ligand that interacts with a predetermined analyte is formed on the surface of the metal film;
A measurement chip comprising: a second region that is provided adjacent to the first region and has a reflectance that serves as a reference for the light having the wavelength and the incident angle. In the first region, the substrate is covered with a metal film having an optimum thickness for causing surface plasmon resonance when light having the predetermined wavelength and incident angle is incident.
In the second region, the base is not covered with a metal film, or is coated with a metal film having a thickness different from that of the metal film formed in the first region. The measurement chip according to claim 5, wherein the two regions are covered with a fixed film having a uniform thickness. The substrate is coated with a metal film having an optimum thickness for generating surface plasmon resonance when light of the predetermined wavelength and incident angle is incident,
In the first region, an immobilization film is formed on the surface of the metal film,
The measurement chip according to claim 5, wherein the surface of the metal film is not covered with an immobilization film in the second region.