JP2010091321A - Substrate for bio-sensing - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a substrate for bio-sensing having new structure which causes large variation in reflectance when protein, DNA and the like are combined with or adsorbed to the surface of the substrate. <P>SOLUTION: The substrate 10 for bio-sensing includes: a substrate lower layer 5 composed of a metal or an alloy; a dielectric thin film 4 formed on the substrate lower layer 5; and a metal thin film or an alloy thin film 3, formed on the dielectric thin film 4, which causes abnormal reflection. Using the abnormal reflection allows bio-sensing to be done with simple reflection to reduce the cost of a bio-sensing chip and a detecting system thereof. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

  The present invention relates to a biosensing substrate. Specifically, the present invention relates to a highly sensitive biosensing substrate using a metal insulator metal (MIM) structure.

FIG. 13 is a diagram for explaining the principle of biosensing. In biosensing such as DNA and protein, a ligand 6 having an interaction specific to the molecule 2 to be detected (analyte) 2 is coated on a substrate 7 such as gold or fine particles, and the binding of the analyte 2 to the ligand 6 is performed. Detect the presence or absence. The binding amount of the analyte 2 is 1 ng / mm ² or less, which is very small. The reflectance change accompanying the coupling is small, and it is difficult to detect it by a normal optical method. Therefore, sensitization has been performed using a surface plasmon resonance (SPR) apparatus, a localized plasmon resonance (LPR) in a gold nanoparticle, or the like.

FIG. 14 is a diagram for explaining biosensing using SPR. FIG. 14A shows a measurement method based on the attenuated total reflection (ATR) method using SPR (hereinafter referred to as ATR-SPR). In ATR-SPR, propagation type surface plasmons (SPs) generated in the metal thin film 12 deposited on the bottom surface of the prism 11 are excited. FIG. 14B shows the dependency of the light reflectance on the incident angle θ. The reflectance of the p-polarized light is minimized at the resonance angle θr, and the energy of the incident light 13 is converted into SPs in the thin film. The reflectance is obtained from the reflected light 14. Since the resonance angle θr is sensitive to the refractive index in the vicinity of the metal thin film 12 and the presence or absence of a substance, if the surface is modified with the ligand 6 in advance, the binding of the analyte 2 can be read as a change in θr. Up to 12% change can be obtained with ATR-SPR due to the binding of 1 ng / mm ² molecules in air. Many biosensors using this method are already on the market and are now indispensable tools in the fields of biochemistry and genetic engineering.

  On the other hand, LPR is SPs excited by gold nanoparticles or a rough metal surface. Unlike ATR-SPR, in order to excite LPR, light may be irradiated to the fine particles. Since there is no need to enter light at a specific incident angle, there is no need to construct an optical system, and there is an advantage that sensing can be performed by measuring transmission, reflection, and scattering spectra using a commercially available spectroscope.

  Under these circumstances, the inventors were able to detect DNA and proteins by simple reflectance measurement using the fact that the reflectance of the gold surface with respect to blue and violet light was 50% or less under several years ago. It showed that. Gold is a metal, but optically exhibits dielectric properties for blue and violet light. Therefore, the reflectance with respect to blue or purple light is about 50%, and therefore it has a yellowish color. The reflectivity of the gold surface with respect to light of these wavelengths is relatively reduced with the adsorption and bonding of substances. This is called an anomalous reflection (AR) of gold. When AR is used, there is a possibility that the gold surface in the air can be changed in reflectivity due to adsorption and binding of substances with respect to light having a wavelength of 470 nm. This phenomenon does not occur in the case of metals such as silver and aluminum. AR may be perpendicular incidence or oblique incidence, and there is no limitation on the thickness of the gold thin film. Therefore, it is possible to measure the amount of trace substances bound to and adsorbed on the gold surface by simple reflectance measurement at normal incidence. Since monochromaticity of incident light is not a serious problem, an incoherent light source such as a light emitting diode (LED) can be used, and therefore, spectroscopic measurement is easy. When gold is used, the sensitivity is unsatisfactory compared to SPR, but various uses are possible because a simple optical system is used. (See Non-Patent Document 1)

S. Watanabe, K.M. Usui, K .; -Y. Tomizaki, K.K. Kajikawa, H .; Mihara, “Anomalous Reflection of Gold Applicable for a Practical Protein-Detecting Chip Platform” Mol. BioSystems 1 (2005) 363-365.

  However, when ATR-SPR is used, a fixed optical system is required, the total reflection attenuation method must be used, and there are limitations on combinations with other measurement methods, and the gold film thickness is 45. There are many points that need to be improved, for example, it is difficult to measure with high sensitivity unless it is between −55 nm, and there is a problem that it is not easy to solve these points. In addition, when LPR is used, there is a problem that a calibration curve needs to be prepared in advance in order to perform quantitative measurement, and sensitivity correction is necessary depending on the size of the molecule.

The inventors obtained the dielectric constant of an AR substrate using abnormal reflection with high sensitivity by simulation, and changed the dielectric constant of the substrate by mixing gold and other substances (silver, etc.) to provide a substrate with high sensitivity. (Patent application 2007-223950) (hereinafter referred to as a comparative example). As a result, when the gold-silver alloy thin film is treated with UV-ozone, an abnormal reflectance change of 1.3% is obtained due to the bonding of molecules of 1 ng / mm ^{2 in} the air. An improved substrate could be developed. Thereby, it was possible to provide a measurement using abnormal reflection, replacing the measurement using the conventional ATR-SPR or the measurement using LPR.

  The present invention aims to develop a highly sensitive substrate structure for an AR substrate using abnormal reflection as compared with a substrate structure made of a single medium as in the comparative example.

  An object of the present invention is to provide a biosensing substrate having a new structure that gives a large change in reflectance when protein, DNA, or the like is bound to or adsorbed to the substrate surface.

  In order to solve the above problems, a biosensing substrate 10 according to the first aspect of the present invention is formed on a substrate lower layer 5 made of a metal or an alloy and a substrate lower layer 5 as shown in FIG. A dielectric thin film 4 and a metal thin film or alloy thin film 3 formed on the dielectric thin film 4 and causing abnormal reflection.

  Here, the substrate lower layer 5 is a substrate component for forming the dielectric thin film 4 and the metal thin film or alloy thin film 3 thereon, and the substrate lower layer 5 is formed on a support plate such as a glass chip or a plastic chip. May be. Abnormal reflection is a metal or alloy that exhibits dielectric properties for optically blue or violet light, such as gold, and the reflectivity for blue or violet light decreases. This is a phenomenon in which the reflectivity decreases relatively with the adsorption and binding of substances. When configured in this manner, it is possible to provide a biosensing substrate having a new structure that gives a large change in reflectance when protein, DNA, or the like is bound to or adsorbed to the substrate surface. Further, by using abnormal reflection, biosensing can be performed with simple reflection, and the cost of the biosensing chip and its detection system can be reduced. This may replace the existing biosensing method using surface plasmon resonance.

  Further, in the biosensing substrate 10 according to the second aspect of the present invention, the metal thin film or the alloy thin film 3 is mainly composed of gold or copper in the first aspect. If comprised in this way, since gold | metal | money or copper produces abnormal reflection, the reflectance change by abnormal reflection is realizable.

In addition, in the biosensing substrate 10 according to the third aspect of the present invention, the dielectric thin film 4 is transparent in the first or second aspect. Here, as the transparent dielectric thin film 4, for example, PMMA (polymethyl methacrylate), glass, SiO ₂ , MgF can be used. If comprised like this aspect, a dielectric thin film will act like a resonator, and the reflectance change by abnormal reflection can be enlarged.

In addition, the biosensing substrate 10 according to the fourth aspect of the present invention has a reflectivity with respect to incident light that is detected in any of the first to third aspects as shown in FIG. 3 or FIG. _{R 0} when there is no target molecules 2 and is referred to as R when there, _{R 0} is 0.5% or more, R / _{R 0} is 0.97% or less, or 1.03% or more. If comprised in this way, it is suitable for the biosensing by an abnormal reflection practically.

Further, the biosensing substrate 10 according to the fifth aspect of the present invention is a film of a metal thin film or an alloy thin film 3 as shown in FIG. 4 or FIG. 8, for example, in any of the first to third aspects. The thickness d ₃ (nm) and the thickness d ₄ (nm) of the dielectric thin film 4 are (8, 18), (8, 52), (13, 57) in the (d ₃ , d ₄ ) coordinate system, The first region surrounded by (17, 57), (17, 28), (12, 18), or (8, 63), (8, 72), (12, 72), (12, 63) It is in the 2nd field surrounded by. If comprised in this way, it is suitable for the biosensing by an abnormal reflection practically.

  ADVANTAGE OF THE INVENTION According to this invention, the board | substrate for biosensing of the new structure which gives a big reflectance change when protein, DNA, etc. couple | bond and adsorb | suck to a substrate surface can be provided.

  Embodiments of the present invention will be described below with reference to the drawings.

[First Embodiment]
FIG. 1 shows a configuration example of a biosensing substrate 10 according to the first embodiment of the present invention. The present embodiment shows an example in which the peripheral medium 1 is a combination of a metal-insulator-metal structure, a gold-PMMA-gold thin film, and air. In FIG. 1, the medium 1 is usually air or water, and in the present embodiment, it is air and the refractive index is n ₁ (= 1). Medium 2 is detected molecule biosensing (analyte, DNA or proteins, etc.), regarded as a continuous dielectric medium having a refractive index n _2. Medium 3 is a metal thin film having a refractive index n _3, the refractive index n ₃ becomes complex. Also, its thickness and _{d 3.} The medium 4 is a dielectric thin film, and its refractive index is n ₄ and its thickness is d ₁ . Medium 5 is a substrate layer of refractive index n _5, the refractive index n ₅ becomes complex. The medium 3 and the medium 5 do not need to be the same metal. The medium 3 to the medium 5 constitute the biosensing substrate 10. The substrate 10 has a structure in which a dielectric (insulator) is sandwiched between metals, and is referred to as a metal-insulator-metal (MIM) structure. 13 is incident light, and 14 is reflected light.

In biosensing, adsorption or binding of dielectric molecules as the analyte 2 to the surface of the medium 3 is detected. Since the adsorption and binding of the molecular layer having a thickness d ₂ = 1 nm correspond to the binding of molecules of 1 ng / mm ² when the density is 1, the wavelength dependence of the reflectance change ΔR at this time is calculated as follows: The sensitivity of the biosensing substrate 10 can be obtained. A dielectric refractive index _{n 4} of the thin film as a medium 4 and 1.5, the refractive index _n 3 of the metal as the medium 3 and the medium 5, _{n 5} literature (P.B.Johnson and R.W.Cristy, " The values described in “Optical Constants of Noble Metals”, Phys. Rev. 6 (1972) 4730-4379.) Were used, and values at wavelengths not described were determined by proportional distribution.

FIG. 2 shows an example of the wavelength dependence of the reflectance of the surface of the medium 3 when the medium 3 is gold, the refractive index n ₂ = 1.5, and the medium 2 having a thickness of 1 nm is adsorbed. R ₀ the reflectance before the medium 2 is adsorbed and _bonding, in which the reflectance after adsorption and binding and R, was plotted to simulate the R / R _0. The calculation condition is that the surrounding medium 1 is air (refractive index 1), the medium 3 is a gold thin film as a metal thin film or an alloy thin film, the medium 4 is a dielectric thin film having a refractive index of 1.5, and the medium 5 is a gold substrate lower layer. Simulations were performed for several combinations using the thickness d ₃ of the medium ₃ and the thickness d ₄ of the medium ₄ as parameters. Since the medium 5 is a gold thin film that is sufficiently thick if it is 100 nm or more, it can be considered as semi-infinite in the calculation. FIG. 2A shows an example in which the thickness d ₃ of the medium 3 is 10 nm and the thickness d ₄ of the medium 4 is 35 nm to 70 nm. FIG. 2B shows the thickness d ₃ of the medium 3 is 15 nm. examples of thickness _d 4 = _55nm~68nm, showing an example of a thickness _d 4 = _70nm~80nm in FIG thickness of the (c) the medium 3 _d 3 = 20nm, medium 4.

According to FIG. 2A, when d ₄ = 35 nm, R / R ₀ takes a value of around 95.0% in a wide wavelength region of wavelengths from 400 nm to 500 nm. That is, it can be seen that the reflectance change of 5.0% occurs due to the coupling of the medium 2. Further, when d ₄ = 40 nm, R / R ₀ is 94.7% as a minimum value, when d ₄ = 45 nm, R / R ₀ is 90.9% as a minimum value, and when d ₄ = 50 nm, R / R ₀ is R / R ₀ has a downwardly convex peak with a minimum value of 85.0%, and when d ₄ = 55 nm, it similarly has a downwardly convex peak and R / R ₀ has a minimum value of 64.0%. . Further, when d ₄ = 60 nm, it has a convex peak, and at a wavelength of about 480 nm, R / R ₀ is 163% at the maximum value, and when d ₄ = 65 nm, R / R ₀ = 116% at the maximum value, When d ₄ = 70 nm, the maximum value is R / R ₀ = 109%. given various extremes of R / R ₀ by the value of d _4, a large change in reflectivity is sometimes obtained. However, as will be described later, it is not always preferable in practice to obtain a large change in reflectance. The wavelength showing a minimum or maximum value as the value of d ₄ is increased becomes long. This confirms that the dielectric thin film 4 acts like a resonator.

FIG. 2B shows that when d ₄ = 55 nm, R / R ₀ has a minimum value of 87.0% near the wavelength of 480 nm, and a reflectance change of 13% occurs. _Further, d 4 = _R / R ₀ when the 60nm _74% at the minimum value, d 4 = _R / R ₀ when the 62nm _56% at the minimum value, R / _{R 0} when the d 4 = 63 nm is The minimum value is 32.0%. Further, when d ₄ = 65 nm, there is a convex peak, R / R ₀ is a maximum value of 353%, and similarly, when d ₄ = 66 nm, ΔR / R is a maximum value of 180% and d ₄ = 68 nm. In this case, the maximum value of R / R ₀ is 132%. Various values of R / R ₀ are given by the value of d _{4, and} a large change in reflectance is obtained in any case. However, it is not always preferable in practice to obtain a large change in reflectance. The wavelength showing a minimum or maximum value as the value of d ₄ is increased becomes long.

According to FIG. 2 (c), when d ₄ = 70 nm, R / R ₀ has a sharp peak protruding downward, and the minimum value is 73.0%. It can be seen that a reflectance change of 27% has occurred. Further, when d ₄ = 72 nm, R / R ₀ is 53% as a minimum value, and when d ₄ = 73 nm, R / R ₀ is 24% as a minimum value. When d ₄ = 76 nm, the peak is convex upward, and R / R ₀ has a maximum value of 187%. When d ₄ = 78 nm, R / R ₀ has a maximum value of 140%, and when d ₄ = 80 nm. R / R ₀ is a maximum value of 125%. given different [Delta] R / R by the value of d _4, a large reflectance change in either case can be obtained. However, it is not always preferable in practice to obtain a large change in reflectance. Further, as the value of d ₄ increases, the wavelength indicating the minimum value or the maximum value becomes longer. When comparing FIGS. 2A to 2C, as the value of d ₃ increases, the same d _{4 is} obtained. The wavelength indicating the minimum value or the maximum value also becomes longer. This confirms that the gold thin film 3 also contributes to the action of the resonator.

One index representing the sensitivity of the biosensing substrate is R / _R0 , which is required to be sufficiently smaller or larger than 1 when the medium 2 is adsorbed and bonded. In the abnormal reflection of gold, R / R ₀ is about 98.7% in air. As shown in FIG. 2, a large change can be obtained in the MIM substrate. However, even if the substrate gives such a large R / R ₀ , if the reflectance R ₀ with respect to the incident light 13 is small, the obtained signal is small and a good result cannot be obtained.

FIG. 3 shows a plot of the relationship between R / R ₀ and R ₀ for the combination of d ₃ and d ₄ described above when the medium 3 is gold and the surrounding medium 1 is air. ● is data of d ₃ = 10 nm, □ is data of d ₃ = 15 nm, ◇ is data of d ₃ = 20 nm, and the value of R / R ₀ for each d ₄ (nm) is plotted as a maximum value or a minimum value. For example, when d ₄ = 40 nm, R / R ₀ = 94.7%, d ₄ = 55 nm, R / R ₀ = 64.0%, d ₄ = 65 nm, R / R ₀ = 116%, and d ₄ = 70 nm. R / R ₀ = 109%. When an incoherent light source such as a halogen lamp is used, a reflectance of at least about 0.5% of the incident light intensity is required in order to ensure a sufficient S / N ratio by measurement for about 1 second. Therefore, even if R / R ₀ is sufficiently smaller than 1 or sufficiently large, if R ₀ <0.5%, the reflectance is too low and it is difficult to say that it is suitable as a biosensing substrate. Moreover, if the amount of change when the medium 2 having a thickness of 1 nm is adsorbed / coupled to the medium 3 is 3% or more, the substrate for abnormal sensing and the biosensing of the comparative example (reflectance change 1.3%) is a prior art. The area that satisfies these two conditions is indicated by hatching.

4, the thickness _d 3 of the thin metal film or alloy film 3 (nm) and the thickness _d 4 of the dielectric thin film 4 (nm) _is, in _(d 3, _{d 4)} coordinate system, as a substrate for the biosensing A combination in a region that satisfies an appropriate condition is indicated by line I. From this, it was found that when the medium 3 is gold and d ₃ = 10 nm in the air, d ₄ = 20 to 50 nm or 65 to 70 nm is preferable. It was also found that d ₄ = 30 to 55 nm gives good results when d ₃ = 15 nm. When the margin is included, the first region (FIG. 18) surrounded by (8, 18), (8, 52), (13, 57), (17, 57), (17, 28), (12, 18). In the region 1) or in the second region (indicated as region 2 in the figure) surrounded by (8, 63), (8, 72), (12, 72), (12, 63). Some points are presumed to give good results. In other cases, R is small, and in order to put it to practical use, it is necessary to produce a highly sensitive detection device and an ideal structure. Note that measurement points in FIG. 5C described later are included in the first region as indicated by x.

  Next, a measurement example of biosensing in the present embodiment will be described. Medium 3? The substrate 10 having the MIM structure constituted by the medium 5 is manufactured, and the reflectance is measured to perform biosensing. When bio-derived molecules as the medium 2 are bound to the substrate 10, a change in reflectance occurs, and this change in reflectivity is monitored by an apparatus including a spectroscope and a detector to detect bio-derived molecules. Since biosensing can be performed with simple reflection using a simple optical system, the cost of the biosensing chip and its detection system can be reduced.

  First, a gold thin film as a metal thin film or alloy thin film of the medium 3, a PMMA (polymethyl methacrylate) thin film as a dielectric thin film of the medium 4, and spin coating on gold as a substrate lower layer of the medium 5, the biosensing substrate 10. A MIM substrate was prepared.

FIG. 5 shows a measurement example of the reflection / absorption spectrum when the medium 2 is adsorbed and bonded to the MIM substrate 10. FIG. 5A shows an example in which the thickness d ₃ of the gold thin film is 10 nm and the thickness d ₄ of the PMMA thin film is 20 nm. (1) (1 in circles) is an AUT self-assembled monolayer film as the first layer of medium 2 prepared by immersing in an ethanol solution of AUT (aminoundecanethiol) for 1 hour and rinsing ( The reflection absorption spectrum of (SAM) is shown. Measurements were made at normal incidence. It is known from the measurement of SPR and ellipsometry that AUT SAM works as a dielectric thin film having a thickness of 1.4 nm and a refractive index of 1.5. The thickness d ₄ of the PMMA thin film as a medium 4 can be known by comparing the results with the simulation results of the measured reflection absorption spectrum (AR spectrum), thereby, the thickness of the PMMA film d ₄ = 20 nm could be estimated. In addition, (2) (2 in circles in the figure) shows a reflection absorption spectrum when the substrate on which AUT SAM is formed is biotinylated with Biotin-OSU as the second layer of the medium 2. (3) (3 in the circle in the figure) indicates that the biotinylated substrate is further exposed to anti-biotin antibody (IgG) (40 μM in PBS) as the third layer of medium 2 for 1 hour, rinsed with PBS and dried. The reflection / absorption spectrum is shown. Thus, it is understood that the reflection / absorption spectrum changes by adsorbing and coupling the medium 2 to the MIM substrate 10.

FIG. 5B shows an example in which the thickness d ₃ = 10 nm of the gold thin film and the thickness d ₄ = 55 nm of the PMMA thin film. This is an example of the result of giving a high reflectance change, and the reflectance change is large but outside the region of FIG. As in the above (1) (1 in circle in the figure) is when AUT SAM is formed, (2) (2 in circle in the figure) is biotinylated, (3) (3 in circle in the figure) ) Is the result when IgG was bound. A large change in reflectance is obtained in the AUT, but the amount of change due to the binding of IgG is small. This is thought to be because if the sensitivity is too high, it will be saturated.

FIG. 5C shows an example in which the thickness d ₃ of the gold thin film is 12 nm and the thickness d ₄ of the PMMA thin film is 55 nm. This is an example of measurement using a substrate with a slightly lower sensitivity than in FIG. The reflectance change is large and is within the first region of FIG. As in the above (1) (1 in circle in the figure) is when AUT SAM is formed, (2) (2 in circle in the figure) is biotinylated, (3) (3 in circle in the figure) ) Is the result when IgG was bound. It can be seen that the binding by IgG is also observed well.

  As described above, according to the present embodiment, it is possible to provide a biosensing substrate having a new structure that gives a large change in reflectance when protein, DNA, or the like is bound to or adsorbed to the substrate surface. Also, by using abnormal reflection, biosensing can be performed with simple reflection, and the cost of the biosensing chip and its detection system can be reduced. This may be replaced with a biosensing method using existing surface plasmon resonance or localized surface plasmon resonance.

[Second Embodiment]
In the second embodiment, a case where the medium 3 is gold and the surrounding medium is water (refractive index n ₁ = 1.33) will be described. In-situ observation of biosensing mainly involves detection in water or buffer solution, so consider the case where the surrounding medium is water. The structure of the substrate is an MIM substrate as in the first embodiment.

FIG. 6 shows the wavelength dependency of the reflectance of the surface of the medium 3 when the medium ₂ having a refractive index n ₂ = 1.5 and a thickness of 1 nm is adsorbed when the surrounding medium is water. The calculation condition is different from the first embodiment only in the refractive index of the surrounding medium 1 (water is 1.33). FIG. 6A shows an example in which the thickness d ₃ of the medium 3 is 10 nm and the thickness d ₄ of the medium 4 is 35 nm to 70 nm. FIG. 6B shows the thickness d ₃ of the medium 3 is 15 nm. examples of thickness _d 4 = _60nm~70nm, the thickness of the medium 3 in FIG. 6 (c) shows an example of a _d 3 = 20 nm, the thickness of the medium 4 _d 4 = 68nm~75nm.

FIG. 6A shows that R / R ₀ is 95.5% and a reflectance change of 4.5% occurs when d ₄ = 35 nm in a wide region having a wavelength of 400 nm or less. _Further, d 4 = _R / R ₀ when the 40nm is _93.9% at the minimum value, the peak of the convex appears below in the vicinity of 370nm when the _{d 4 = 45nm, R / R} 0 is the minimum value 90. When 1% and d ₄ = 50 nm, R / R ₀ has a minimum value of 84.9%, and when d ₄ = 55 nm, the peak has a sharp convex peak, and R / R ₀ has a minimum value of 18.4%. It becomes. Further, when d ₄ = 60 nm, the peak is convex upward and the maximum value is R / R ₀ = 116%. When d ₄ = 65 nm, the maximum value is R / R ₀ = 106% and d ₄ = 70 nm. The maximum value is R / R ₀ = 104%. given various extremes of R / R ₀ by the value of d _4, a large change in reflectivity is sometimes obtained. However, it is not always preferable in practice to obtain a large change in reflectance. The wavelength showing a minimum or maximum value as the value of d ₄ is increased becomes long.

According to FIG. 6B, in the region of wavelength 500 nm or less, when d ₄ = 60 nm, R / R ₀ is 90.2% at the minimum value near the wavelength 475 nm, and the reflectance change is 9.8%. You can see what is happening. Further, when d ₄ = 62 nm, R / R ₀ had a minimum value of 83%, and when d ₄ = 63 nm, R / R ₀ had a minimum value of 71%, and a convex peak appeared. Further, when d ₄ = 65 nm, a convex peak appears, and R / R ₀ has a maximum value of 171%, and when d ₄ = 70 nm, R / R ₀ has a maximum value of 108%. Various values of R / R ₀ are given by the value of d _{4, and} a large change in reflectance is obtained in any case. However, it is not always preferable in practice to obtain a large change in reflectance. The wavelength showing a minimum or maximum value as the value of d ₄ is increased becomes long.

According to FIG. 6C, a convex peak appears downward when d ₄ = 68 nm in the region near the wavelength of 500 nm, and R / R ₀ is 86.9% as a minimum value, and is reflected at 13.1%. It can be seen that a rate change has occurred. Further, when d ₄ = 70 nm, R / R ₀ is 68.2% as a minimum value. Further, when d ₄ = 72 nm, R / R ₀ has a maximum value of 157%, and when d ₄ = 75 nm, R / R ₀ has a maximum value of 110%. Various values of R / R ₀ are given by the value of d _{4, and} a large change in reflectance is obtained in any case. However, it is not always preferable in practice to obtain a large change in reflectance. Further, as the value of d ₄ increases, the wavelength indicating the minimum value or the maximum value becomes longer. When comparing FIGS. 6A to 6C, as the value of d ₃ increases, the same d _{4 is} obtained. The wavelength indicating the minimum value or the maximum value also becomes longer.

FIG. 7 shows a plot of the relationship between R / R ₀ and R ₀ against the combination of d ₃ and d ₄ described above when the medium 1 is water. ● is data of d ₃ = 10 nm, □ is data of d ₃ = 15 nm, ◇ is data of d ₃ = 20 nm, and the value of R / R ₀ for each d ₄ (nm) is plotted as a maximum value or a minimum value. For example, when d ₄ = 40 nm, R / R ₀ = 93.9%, and when d ₄ = 70 nm, R / R ₀ = 104%. In addition, a region suitable for a biosensing substrate is indicated by hatching, where R ₀ is 0.5% or more and the amount of change when the medium 2 having a thickness of 1 nm is adsorbed / bonded to the medium 3 is 3% or more.

8, the thickness _d 3 of the thin metal film or alloy film 3 (nm) and the thickness _d 4 of the dielectric thin film 4 (nm) _is, in _(d 3, d ₄₎ coordinate system, the above condition is satisfied region A combination is indicated by line I. From this, it was found that when the medium 1 is water, d ₄ = 35 to 50 nm or 65 to 70 nm is preferable when d ₃ = 10 nm. Further, it was found that when d ₃ = 15 nm, R ₀ = 0.5% or more and R / R ₀ = 0.95% when d ₄ = 50 to 55 nm, giving good results. When a margin is included, a third region (FIG. 5) surrounded by (8, 33), (8, 52), (13, 57), (17, 57), (17, 48), (12, 33). In this case, it is estimated that a point in the second region surrounded by (8, 63), (8, 72), (12, 72), (12, 63) gives a good result. Is done. Note that the third region is included in the first region, and the first region is also a region that gives good results in either air or water.

  Also in this embodiment, since the MIM structure substrate is used, as in the first embodiment, a biostructure with a new structure that gives a large change in reflectivity when protein or DNA is bound to or adsorbed to the substrate surface. A sensing substrate can be provided. Also, by using abnormal reflection, biosensing can be performed with simple reflection, and the cost of the biosensing chip and its detection system can be reduced.

[Third Embodiment]
In the third embodiment, a case where the medium 5 is silver will be described.
FIG. 9 shows an example of a simulation result of the reflection / absorption spectrum when the medium 5 is silver and the surrounding medium 1 is air. This is an example of the wavelength dependence of the reflectance of the surface of the medium 3 when the medium ₂ having a refractive index n ₂ = 1.5 and a thickness of 1 nm is adsorbed. R ₀ the reflectance before the medium 2 is adsorbed and _bonding, in which the reflectance after adsorption and binding and R, was plotted to simulate the R / R _0. The calculation condition is that the surrounding medium 1 is air (refractive index 1), the medium 3 is a gold thin film as a metal thin film or an alloy thin film, the medium 4 is a dielectric thin film having a refractive index of 1.5, and the medium 5 is a silver substrate lower layer. Simulations were performed for several combinations using the thickness d ₃ of the medium ₃ and the thickness d ₄ of the medium ₄ as parameters. Since the medium 5 is a silver thin film that is sufficiently thick if it is 100 nm or more, it can be considered as semi-infinite in the calculation. FIG. 9A illustrates an example in which the thickness d ₃ of the medium 3 is 10 nm and the thickness d ₄ of the medium 4 is 40 nm to 70 nm. FIG. 9B illustrates the thickness d ₃ of the medium 3 is 15 nm. examples of thickness _d 4 = _40nm~75nm, showing an example of a thickness _d 4 = _40nm~75nm thickness _d 3 = 20 nm of the medium 3 in FIG. 9 (c), the medium 4.

According to FIG. 9A, when d ₄ = 40 nm, R / R ₀ increases by 5% or more in the wavelength region of wavelength from 330 nm to 375 nm, and R / R ₀ increases by 5% or more in the wavelength region of wavelength from 375 nm to 450 nm. Decrease. That is, it can be seen that the reflectance change of 5.0% occurs due to the coupling of the medium 2. When d ₄ = 45 nm, R / R ₀ increases by 5% or more in the wavelength region of wavelengths from 350 nm to 380 nm, and R / R ₀ decreases by 5% or more in the wavelength region of wavelengths from 400 nm to 450 nm. That is, it can be seen that the reflectance change of 5.0% occurs due to the coupling of the medium 2. When d ₄ = 50 nm, R / R ₀ increases by 5% or more in the wavelength region of wavelength 370 nm to 420 nm, and R / R ₀ decreases by 5% or more in the wavelength region of wavelength 430 nm to 450 nm. That is, it can be seen that the reflectance change of 5.0% occurs due to the coupling of the medium 2. When d ₄ = 55 nm, R / R ₀ increases by 5% or more in the wavelength range of 400 nm to 430 nm. That is, it can be seen that the reflectance change of 5.0% occurs due to the coupling of the medium 2. When d ₄ = 60 nm, R / R ₀ increases by 5% or more in the wavelength region of 420 nm to 440 nm. That is, it can be seen that the reflectance change of 5.0% occurs due to the coupling of the medium 2. When d ₄ = 65 nm, R / R ₀ increases by 5% or more in the wavelength range of 430 nm to 440 nm. That is, it can be seen that the reflectance change of 5.0% occurs due to the coupling of the medium 2. However, as will be described later, it is not always preferable in practice to obtain a large change in reflectance. The wavelength showing a minimum or maximum value as the value of d ₄ is increased becomes long. This confirms that the dielectric thin film 4 acts like a resonator.

According to FIG. 9B, when d ₄ = 40 nm, R / R ₀ decreases by 5% or more in the wavelength region of wavelengths from 330 nm to 470 nm. That is, it can be seen that the reflectance change of 5.0% occurs due to the coupling of the medium 2. When d ₄ = 45 nm, R / R ₀ decreases by 5% or more in the wavelength range of 390 nm to 470 nm. That is, it can be seen that the reflectance change of 5.0% occurs due to the coupling of the medium 2. When d ₄ = 50 nm, R / R ₀ decreases by 5% or more in the wavelength range of 410 nm to 470 nm. That is, it can be seen that the reflectance change of 5.0% occurs due to the coupling of the medium 2. When d ₄ = 55 nm, R / R ₀ decreases by 5% or more in the wavelength range of 430 nm to 470 nm. That is, it can be seen that the reflectance change of 5.0% occurs due to the coupling of the medium 2. When d ₄ = 60 nm, R / R ₀ is decreased by 5% or more in the wavelength region of 450 nm to 470 nm. That is, it can be seen that the reflectance change of 5.0% occurs due to the coupling of the medium 2. When d ₄ = 65 nm, R / R ₀ increases by 5% or more in the wavelength range of 430 nm to 470 nm. That is, it can be seen that the reflectance change of 5.0% occurs due to the coupling of the medium 2. When d ₄ = 70 nm, R / R ₀ increases by 5% or more in the wavelength range of 450 nm to 470 nm. That is, it can be seen that the reflectance change of 5.0% occurs due to the coupling of the medium 2. When d ₄ = 75 nm, R / R ₀ increases by 5% or more in the wavelength region of 470 nm to 480 nm. That is, it can be seen that the reflectance change of 5.0% occurs due to the coupling of the medium 2. However, as will be described later, it is not always preferable in practice to obtain a large change in reflectance. The wavelength showing a minimum or maximum value as the value of d ₄ is increased becomes long.

According to FIG. 9C, when d ₄ = 60 nm, R / R ₀ decreases by 5% or more in the wavelength region of wavelength 470 nm to 490 nm. That is, it can be seen that the reflectance change of 5.0% occurs due to the coupling of the medium 2. When d ₄ = 65 nm, R / R ₀ decreases by 5% or more in the wavelength range of 470 nm to 490 nm. That is, it can be seen that the reflectance change of 5.0% occurs due to the coupling of the medium 2. When d ₄ = 70 nm, R / R ₀ decreases by 5% or more in the wavelength region of 480 nm to 490 nm. That is, it can be seen that the reflectance change of 5.0% occurs due to the coupling of the medium 2. When d ₄ = 75 nm, R / R ₀ increases by 5% or more in the wavelength range of 470 nm to 480 nm. That is, it can be seen that the reflectance change of 5.0% occurs due to the coupling of the medium 2. However, as will be described later, it is not always preferable in practice to obtain a large change in reflectance. Further, as the value of d ₄ increases, the wavelength indicating the minimum value or the maximum value becomes longer. When comparing FIGS. 9A to 9C, as the value of d ₃ increases, the same d _{4 is} obtained. The wavelength indicating the minimum value or the maximum value also becomes longer. This confirms that the gold thin film 3 also contributes to the action of the resonator.

FIG. 10 shows an example of a simulation result of the reflection absorption spectrum when the medium 5 is silver and the surrounding medium 1 is water. This is an example of the wavelength dependence of the reflectance of the surface of the medium 3 when the medium ₂ having a refractive index n ₂ = 1.5 and a thickness of 1 nm is adsorbed. R ₀ the reflectance before the medium 2 is adsorbed and _bonding, in which the reflectance after adsorption and binding and R, was plotted to simulate the R / R _0. The calculation conditions are: peripheral medium 1 is water (refractive index 1.33), medium 3 is a gold thin film as a metal thin film or alloy thin film, medium 4 is a dielectric thin film having a refractive index of 1.5, and medium 5 is a silver substrate lower layer. The simulation was performed for several combinations using the thickness d ₃ of the medium ₃ and the thickness d ₄ of the medium ₄ as parameters. Since the medium 5 is a silver thin film that is sufficiently thick if it is 100 nm or more, it can be considered as semi-infinite in the calculation. FIG. 10A shows an example in which the thickness d ₃ of the medium 3 is 15 nm and the thickness d ₄ of the medium 4 is 15 nm to 40 nm. FIG. 10B shows the thickness d ₃ of the medium 3 is 20 nm. examples of thickness _d 4 = _45nm~70nm, showing an example of a thickness _d 4 = _50nm~80nm of Figure 10 the thickness of the (c) the medium 3 _d 3 = 25nm, medium 4.

According to FIG. 10A, when d ₄ = 30 nm, R / R ₀ decreases by 5% or more in the wavelength region of wavelengths from 340 nm to 360 nm. That is, it can be seen that the reflectance change of 5.0% occurs due to the coupling of the medium 2. When d ₄ = 35 nm, R / R ₀ decreases by 5% or more in the wavelength region of 360 to 380 nm. That is, it can be seen that the reflectance change of 5.0% occurs due to the coupling of the medium 2. When d ₄ = 40 nm, R / R ₀ decreases by 5% or more in the wavelength region of wavelengths from 370 nm to 390 nm. That is, it can be seen that the reflectance change of 5.0% occurs due to the coupling of the medium 2. However, as will be described later, it is not always preferable in practice to obtain a large change in reflectance. The wavelength showing a minimum or maximum value as the value of d ₄ is increased becomes long.

According to FIG. 10B, when d ₄ = 60 nm, R / R ₀ decreases by 5% or more in the wavelength region of wavelengths from 440 nm to 460 nm. That is, it can be seen that the reflectance change of 5.0% occurs due to the coupling of the medium 2. When d ₄ = 65 nm, R / R ₀ decreases by 5% or more in the wavelength region of wavelengths from 450 nm to 470 nm. That is, it can be seen that the reflectance change of 5.0% occurs due to the coupling of the medium 2. When d ₄ = 70 nm, R / R ₀ increases by 5% or more in the wavelength range of 460 nm to 460 nm. That is, it can be seen that the reflectance change of 5.0% occurs due to the coupling of the medium 2. When d ₄ = 75 nm, R / R ₀ increases by 5% or more in the wavelength range of 470 nm to 480 nm. That is, it can be seen that the reflectance change of 5.0% occurs due to the coupling of the medium 2. When d ₄ = 80 nm, R / R ₀ increases by 5% or more in the wavelength region of 480 nm to 485 nm. That is, it can be seen that the reflectance change of 5.0% occurs due to the coupling of the medium 2. However, it is not always preferable in practice to obtain a large change in reflectance. The wavelength showing a minimum or maximum value as the value of d ₄ is increased becomes long.

According to FIG. 10C, when d ₄ = 70 nm, R / R ₀ decreases by 5% or more in the wavelength region of wavelengths 470 nm to 480 nm. That is, it can be seen that the reflectance change of 5.0% occurs due to the coupling of the medium 2. When d ₄ = 80 nm, R / R ₀ decreases by 5% or more in the wavelength range of 480 nm to 485 nm. That is, it can be seen that the reflectance change of 5.0% occurs due to the coupling of the medium 2. When d ₄ = 80 nm, R / R ₀ increases by 5% or more in the wavelength region of 475 nm to 485 nm. That is, it can be seen that the reflectance change of 5.0% occurs due to the coupling of the medium 2. However, as will be described later, it is not always preferable in practice to obtain a large change in reflectance. Further, as the value of d ₄ increases, the wavelength indicating the minimum value or the maximum value becomes longer. When comparing FIGS. 10A to 10C, as the value of d ₃ increases, the same d _{4 is} obtained. The wavelength indicating the minimum value or the maximum value also becomes longer. This confirms that the gold thin film 3 also contributes to the action of the resonator.

One index representing the sensitivity of the biosensing substrate is R / _R0 , which is required to be sufficiently smaller or larger than 1 when the medium 2 is adsorbed and bonded. In the abnormal reflection of gold, R / R ₀ is about 98.7% in air. As shown in FIG. 2, a large change can be obtained in the MIM substrate. However, even if the substrate gives such a large R / R ₀ , if the reflectance R ₀ with respect to the incident light 13 is small, the obtained signal is small and a good result cannot be obtained.

FIG. 11 shows a plot of the relationship between R / R ₀ and R ₀ for the combination of d ₃ and d ₄ described above when the medium 5 is silver. FIG. 11A shows an example where the surrounding medium 1 is air, and FIG. 11B shows an example where the surrounding medium 1 is water. In FIG. 11A and FIG. 11B, ● is data of d ₃ = 10 nm, □ is data of d ₃ = 15 nm, ◇ is data of d ₃ = 20 nm, and R / R ₀ of each d ₄ (nm) Values were plotted as maximum or minimum values. When an incoherent light source such as a halogen lamp is used, a reflectance of at least about 0.5% of the incident light intensity is required in order to ensure a sufficient S / N ratio by measurement for about 1 second. Therefore, even if R / R ₀ is sufficiently smaller than 1 or sufficiently large, if R ₀ <0.5%, the reflectance is too low and it is difficult to say that it is suitable as a biosensing substrate. Further, if the amount of change when the medium 2 having a thickness of 1 nm is adsorbed and bonded to the medium 3 is 3% or more, it is considered that it is superior to the prior art abnormal reflection and the biosensing substrate of the comparative example. Therefore, areas that satisfy these two conditions are indicated by hatching.

FIG. 12 shows combinations in a region satisfying conditions suitable as a biosensing substrate in the (d ₃ , d ₄ ) coordinate system when the medium 5 is silver. FIG. 12A shows the case where the surrounding medium 1 is air, and FIG. 12B shows the case where the surrounding medium 1 is water. Thickness _d 3 of the thin metal film or alloy film 3 (nm) and the thickness _d 4 of the dielectric thin film 4 (nm) _is, in _(d 3, d ₄₎ coordinate system, is suitable as a substrate for the biosensing conditions A combination in a region satisfying is indicated by a line I. FIG. 12A shows that d ₄ = 40 to 65 nm is preferable in the air when d ₃ = 10 nm. It was also found that d ₄ = 40 to 75 nm gives good results when d ₃ = 15 nm. It was also found that d ₄ = 60 to 75 nm gives good results when d ₃ = 20 nm. Including the margin, (8, 52), (8, 72), (13, 77), (17, 77), (22, 62), (22, 48), (17, 43), (13, 38) and the fifth region surrounded by (12, 68), (18, 68), (18, 52), (12, 52) in the fourth region (shown as region 4 in the figure). It is estimated that a point in a portion excluding the region (shown as region 5 in the figure) gives a good result. In other cases, R is small, and in order to put it to practical use, it is necessary to produce a highly sensitive detection device and an ideal structure.

Moreover, from 12 (b), it was found that d ₄ = 30 to ₄₀ nm is preferable in water when d ₃ = 15 nm. It was also found that d ₄ = 60 to 70 nm gives good results when d ₃ = 20 nm. It was also found that d ₄ = 75 to 80 nm gives good results when d ₃ = 25 nm. When the margin is included, (13, 13), (13, 32), (23, 77), (27, 77), (27, 72), (27, 58), (22, 42), (18, 43), (17, 57), (23, 57), (23, 73), (17, 73) in the sixth area (shown as area 6 in the figure) surrounded by (17, 13) It is presumed that a point in a portion excluding the seventh region (indicated by region 7 in the figure) surrounded by 与 え る gives a good result. That is, good sensitivity can be obtained even in water under almost the same conditions as in air. In other cases, R is small, and in order to put it to practical use, it is necessary to produce a highly sensitive detection device and an ideal structure.

  Also in this embodiment, since the MIM structure substrate is used, as in the first and second embodiments, a new reflectance change that greatly changes when protein, DNA, or the like is bound to or adsorbed to the substrate surface. A substrate for structure biosensing can be provided. Also, by using abnormal reflection, biosensing can be performed with simple reflection, and the cost of the biosensing chip and its detection system can be reduced.

  Although the embodiments of the present invention have been described above, the present invention is not limited to the above-described embodiments, and it is obvious that various modifications can be made to the embodiments.

For example, in the above embodiment, an example in which the medium 5 is gold has been described, but other metals or alloys may be used. The substrate lower layer 5 may be formed on a support plate such as a glass chip or a plastic chip. In addition, although the example in which the medium 4 is PMMA has been described, other transparent dielectric materials such as glass, SiO ₂ , and MgF may be used. Further, the example in which the medium 3 is gold has been described, but other metals or alloys that cause abnormal reflection, for example, an alloy mainly composed of gold or a metal or alloy mainly composed of copper may be used. The medium 2 can be selected as appropriate, and the medium 1 may be a gas or liquid other than air or water corresponding to the medium 2. Further, the film thickness and refractive index of each medium can be appropriately selected and adjusted.

  The present invention is used for biosensing.

It is a figure which shows the structural example of the board | substrate for biosensing in this invention. When the medium 5 is gold and the surrounding medium is air, it is a figure which shows the example of the wavelength dependence of the reflectance of the medium 3 surface when the medium _{2 of} refractive index n2 = 1.5 adsorb | sucks. Medium 5 is gold, the case surrounding medium 1 is air, is a plot of relationship between the R / R ₀ and R ₀ to the combination of d ₃ and d _4. Medium 5 is gold, if the peripheral medium 1 is air, in _{(d 3,} d ₄₎ coordinate system is a diagram showing a combination in satisfying region it is suitable as a substrate for biosensing. It is a figure which shows the example of a measurement of a reflection absorption spectrum when a medium is formed in a MIM board | substrate. Medium 5 is gold, if the peripheral medium is water, a medium 2 of refractive index n _{2 =} 1.5 is a diagram showing an example of a wavelength dependence of the reflectance of the medium 3 surface when adsorbed. Medium 5 is gold, the case surrounding medium 1 is water, is a plot of relationship between the R / R ₀ and R ₀ to the combination of d ₃ and d _4. Medium 5 is gold, if the peripheral medium 1 is water, the _{(d 3,} d ₄₎ coordinate system is a diagram showing a combination in satisfying region it is suitable as a substrate for biosensing. It is a figure which shows the example of the simulation result of the reflection absorption spectrum in case the medium 5 is silver and the surrounding medium is air. It is a figure which shows the example of the simulation result of the reflection absorption spectrum in case the medium 5 is silver and the surrounding medium is water. If the medium 5 is silver, it is a plot of relationship between the R / _{R 0} and _{R 0} to the combination of _{d 3} and _{d 4.} If the medium 5 is silver, the _{(d 3,} d ₄₎ coordinate system is a diagram showing a combination in satisfying region is suitable as a substrate for biosensing. It is a figure for demonstrating the principle of biosensing. It is a figure for demonstrating the biosensing using surface plasmon resonance.

Explanation of symbols

1 Peripheral medium (medium 1)
2 Molecules to be detected (analyte, medium 2)
3 Metal thin film (medium 3)
4 Dielectric thin film (medium 4)
5 Lower layer of substrate (medium 5)
6 is a refractive index R medium 2 having a thickness of _n 1 ~n ₅ medium 1 medium 5 ligand 7 substrate 10 biosensing substrate 11 prism 12 metal films 13 incident light 14 reflected light _d 2 to d ₄ medium 2 medium 4 Reflectivity R ₀ when present Reflectivity SPs when medium 2 is not present Propagation type surface plasmon ΔR Reflectivity change θ Incident angle θr Resonance angle

Claims (5)

A substrate lower layer made of metal or alloy;
A dielectric thin film formed on the lower layer of the substrate;
A metal thin film or an alloy thin film that is formed on the dielectric thin film and causes abnormal reflection;
Substrates for biosensing. The metal thin film or alloy thin film is mainly composed of gold or copper;
The biosensing substrate according to claim 1. The dielectric thin film is transparent;
The biosensing substrate according to claim 1 or 2. Reflectance of incident light and the absence of the detection target molecule _{R 0,} a and R when there, _{R 0} is 0.5% or more, R / _{R 0} 0.97% or less, or 1.03% Or more;
The biosensing substrate according to any one of claims 1 to 3. The metal thin film or the alloy film thickness _d 3 (nm) and the thickness _d 4 of the dielectric thin film (nm) _is, in _(d 3, _{d 4)} coordinate system, (8,18), (8,52) , (13, 57), (17, 57), (17, 28), (12, 18) surrounded by the first region, or (8, 63), (8, 72), (12, 72) ), In the second region surrounded by (12, 63);
The biosensing substrate according to any one of claims 1 to 3.