JP4759732B2 - DNA detection method, DNA detection metal structure and DNA detection apparatus - Google Patents

Description

  The present invention relates to a sensing method and a sensing device, and more particularly to a sensing method and a sensing device used for medical diagnosis, genetic analysis, and environmental analysis.

  In recent years, in order to perform medical diagnosis, genetic analysis, and environmental analysis quickly, efficiently, and simply, a technology for detecting a very small amount of biologically relevant substances or environmental harmful substances with high sensitivity has become important.

  One of the most promising principles of the technology for detecting the above-mentioned biologically relevant substances or environmental harmful substances is to use a special optical response phenomenon called “localized (surface) plasmon resonance”. Localized plasmon resonance refers to the shape and size of a fine metal structure, such as a metal having a nanoscale surface structure or a nanometer-sized metal fine particle, when irradiated with light having a specific resonance wavelength. This is an optical response in which a local electric field is formed in a wavelength region corresponding to. The local electric field formed by the localized plasmon resonance efficiently excites only the photoresponsive molecule fixed on the surface of the metal structure.

  As the type of metal constituting the metal structure, noble metals such as gold, silver, and platinum are often used, but even if the type of metal is the same, if the shape or size is different, the absorption spectrum wavelength of localized plasmon resonance will be increased. Different. In addition, the intensity of the optical response (luminescence or Raman spectroscopy) of the molecule adsorbed on the metal exhibiting localized plasmon resonance is remarkably enhanced by the interaction between the molecule and plasmon. That is, the metal structure exhibiting localized plasmon resonance functions as a highly sensitive sensor device for a molecular system.

  Examples of medical diagnosis, gene analysis, and environmental analysis using the above principle include DNA analysis using a DNA chip and antigen-antibody diagnosis using an immunoassay chip.

  DNA analysis is a probe molecule having a base sequence complementary to a sample DNA by introducing a sample DNA solution to be detected onto a DNA chip on which a plurality of probe DNAs (single strands) having different base sequences are fixed. Sample DNA is detected by reading the presence or absence of hybridization for each probe DNA (hereinafter referred to as “hybridization”).

  On the other hand, in antigen-antibody diagnosis, a specimen solution is introduced onto an immunoassay chip on which an antibody made by an animal using the detection target substance as an antigen is immobilized, and the antigen in the specimen solution and the antibody on the immunoassay chip are selectively used. The antigen is detected by detecting the complex formed in the cell.

  In such DNA analysis and antigen-antibody diagnosis, it is proposed to sense the detection target substance (fluorescence sensing) by irradiating a sample labeled with a fluorescent molecule with excitation light and detecting the amount of excited fluorescence. Has been.

  However, in fluorescence sensing, there is a serious problem that not only a detection target substance but also a fluorescent impurity (interfering substance) in a sample is simultaneously fluorescent, and the sensitivity and accuracy of sensing are lowered.

  Thus, another sensor that uses a metal structure exhibiting localized plasmon resonance but does not use fluorescence sensing has been proposed that senses a detection target substance using light reflection (scattering) (for example, a patent). Reference 1 and Patent Document 2).

  The surface plasmon resonance sensor described in Patent Document 1 is the following sensor. That is, a light source (organic EL element) and a detector (photodiode) are provided on one surface of a prism (semiconductor silicon) having surfaces parallel to the top and bottom, and a metal thin film (gold film) is provided on the other surface, A light absorbing member (light absorbing block) is provided inside. The light absorbing member blocks the light emitted from other angles from the light source so that the light emitted from the light source at a predetermined angle is incident on the metal thin film and totally reflected to reach the detector. However, the intensity of reflected light is detected.

Further, the localized surface plasmon sensing device described in Patent Document 2 is the following device. That is, a metal fine particle layer having a size capable of exciting the surface plasmon resonance localized on the end face of the optical fiber is formed, and a molecular layer of a molecule complementary to the molecule to be detected is formed on the surface of the metal fine particle layer. Then, using a change in light input to the optical fiber by surface plasmon resonance localized in the metal fine particle layer, detection target molecules adsorbed or bonded to complementary molecules are detected.
JP 2005-156415 A JP 2005-181296 A

  However, in the surface plasmon resonance sensor described in Patent Document 1, the reflected light detected by the detector due to factors such as deviation / blur of the optical arrangement including the optical axis, scratches on the prism, distortion of the prism, or dirt on the mirror surface. The intensity of fluctuates. Therefore, there is a problem that the intensity of reflected light to be detected is not an inherent physical quantity in a state to be detected, but measurement sensitivity and measurement accuracy are lowered.

  Furthermore, also in the localized surface plasmon sensing device described in Patent Document 2, the detection target molecule is detected by detecting the intensity of the reflected light, so that the detection of the detection target molecule is a qualitative detection. There is a problem that it is difficult to detect.

  The present invention has been made in view of the above points, and provides a sensing method and a sensing device capable of detecting a detection target substance with high accuracy and quantitatively without using fluorescence sensing or reflected light. Objective.

  In the sensing method according to the present invention, a detection target substance attached to the metal structure is detected using an absorption spectrum of the localized plasmon resonance induced in the metal structure exhibiting localized plasmon resonance.

  In addition, the sensing method according to the present invention includes a measurement step of measuring an absorption spectrum of localized plasmon resonance induced in a metal structure exhibiting localized plasmon resonance, and a complementary to the detection target substance on the metal structure. Absorption spectrum of localized plasmon resonance measured with one end of the substance fixed, and localization measured with a sample containing the substance to be detected fixed to the complementary structure on the metal structure A calculation step of comparing the absorption spectrum of plasmon resonance and calculating a shift amount of the absorption spectrum; and a detection step of detecting a detection target substance contained in the sample based on the calculated shift amount. I made it.

  In addition, the sensing device according to the present invention includes a metal structure that exhibits localized plasmon resonance, a measurement unit that measures an absorption spectrum of the localized plasmon resonance induced in the metal structure, and a detection on the metal structure. Absorption spectrum of localized plasmon resonance measured with one end of a substance complementary to the target substance fixed, and a sample containing the target substance to be detected fixed to the complementary substance on the metal structure And comparing with the absorption spectrum of the localized plasmon resonance measured in the state, and calculating the shift amount of the absorption spectrum, and based on the calculated shift amount, the detection target substance contained in the sample And a detecting means for detecting.

  According to the present invention, a detection target substance can be detected with high accuracy and quantitativeness without using fluorescence sensing or light reflection.

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

(Embodiment 1)
The present inventor needs to perform sensing using a specific physical quantity in a state to be detected in order to detect a detection target substance with high accuracy and quantitatively without using fluorescence sensing or light reflection. I found. In addition, in order to perform sensing using the specific physical quantity of the state to be detected, the absorption spectrum of the localized plasmon resonance measured with the detection target substance attached on the metal structure showing the localized plasmon resonance is obtained. It was found that it was necessary to use.

  The present invention is a metal structure device (metal nanorod array) in which a large number of metal nanostructures (for example, metal nanorods or metal nanoblocks) are integrated on a solid transparent substrate such as glass by applying semiconductor microfabrication technology. Alternatively, a metal nanoblock array is constructed, and a detection target substance and a substance complementary to the detection target substance (hereinafter abbreviated as “complementary substance”) are fixed on the metal structure device and induced in the metal structure. By detecting the amount of shift in the absorption spectrum of the localized plasmon resonance, the detection target substance attached to the metal structure is detected by detecting the binding between the target substance and the complementary substance on the metal structure. To do.

  Here, since the shift amount of the absorption spectrum is very small, a measurement error is likely to occur. Therefore, the measurement accuracy can be improved by increasing the shift amount of the absorption spectrum of the localized plasmon resonance under the same conditions.

  In order to further increase the shift amount of the absorption spectrum of localized plasmon resonance under the same conditions, the present inventor has determined the size, shape and directionality of the metal nanostructure constituting the metal structure exhibiting localized plasmon resonance. As well as the distance between the metal nanostructures, it has been found that it is necessary to arrange them precisely and regularly.

  In the present specification, “complementary” means a relationship in which a certain substance specifically binds to a specific substance.

  In the present specification, the “metal structure” means, in a narrow sense, a component of a certain metal assembly such as a metal nanorod and a metal nanoblock, and in a broad sense, a metal nanorod array and a metal nanoblock array. It means an aggregate of metal structures in a narrow sense. Furthermore, in the broadest sense, it means a metal structure including a substrate on which a broad sense metal structure is constructed.

  Hereinafter, metal nanorods and / or metal nanoblocks are abbreviated as “metal nanorods (blocks)”, and metal nanorod arrays and / or metal nanoblock arrays are abbreviated as “metal nanorods (blocks) arrays” as appropriate.

  In the present specification, the “aspect ratio” means the ratio of the length of the major axis to the length of the minor axis of the metal nanorod (block). For example, the aspect ratio of a metal nanorod (block) having an anisotropic shape with an average length of the short axis of 10 nm and an average length of the long axis of 100 nm is 10.

  FIG. 1 is a diagram showing an overall configuration of a sensing device according to Embodiment 1 of the present invention. FIG. 2 is a block diagram showing the configuration of the sensing device shown in FIG.

  The sensing device 100 according to the present embodiment broadly includes a metal structure 110, a substrate transfer device 120, a stage 130, an absorption spectrum measuring device 140, a computer unit 150, an input unit 160, and an output unit 170.

  The metal structure 110 is obtained by integrating metal nanostructures (for example, gold, platinum, silver, etc.) with precisely controlled sizes, shapes and orientations, and distances between the metal nanostructures, on a solid transparent substrate. Is. One end of a complementary material is fixed on the surface of the metal structure 110, and after the sample containing the detection target material is introduced, the substrate transfer device 120 is loaded. In addition, the manufacturing method of the metal structure 110 is demonstrated in detail later.

  As described above, the complementary substance is a substance that specifically binds to the detection target substance. For example, in the antigen-antibody reaction, the substance to be detected is an antigen, and the complementary substance is an antibody. In DNA hybridization, the detection target substance is sample DNA, and the complementary substance is probe DNA. Further, when the detection target substance is an antigen, the metal structure 110 becomes a so-called immunoassay chip, and when the detection target substance is DNA, the metal structure 110 becomes a so-called DNA chip.

  For example, one end of a plurality of different antibodies 102 can be fixed to the immunoassay chip (see FIG. 3A). Further, when the introduced antigen 104 causes an antigen-antibody reaction with the immobilized antibody 102, the antigen 104 and the antibody 102 selectively form a complex (see FIG. 3B).

For example, one end of a plurality of probe DNAs 106 having different base sequences can be immobilized on the DNA chip (see FIG. 4A). Further, it is known that when the introduced sample DNA 108 is hybridized with the immobilized probe DNA 106, the chain length of the complex (double strand) is shortened (L ₁ > L ₂ ) (FIG. 4 ( B)). Here, L ₁ is the chain length of the probe DNA, and L ₂ is the chain length of the complex (double strand) obtained by hybridization.

  The substrate transfer device 120 loads the metal structure 110, and transfers the loaded metal structure 110 onto the stage 130 according to an instruction from the control unit 152. In addition, the substrate transfer apparatus 120 can be loaded with a plurality of metal structures 110 at the same time in order to realize multi-analyte multi-item sensing.

  The stage 130 places the metal structure 110 transferred from the substrate transfer device 120. The stage 130 is driven by a stage driving device 132 on a plane perpendicular to the optical axis of the probe light emitted in the absorption spectrum measuring device 140.

  The absorption spectrum measuring apparatus 140 roughly includes a light source controller 141, a power source 142, a light source 143, a lens 144, an interferometer 145, a detection unit 146, and an absorption spectrum calculation unit 147. The absorption spectrum measuring apparatus 140 is, for example, a microscopic Fourier transform infrared absorption spectrum wavelength measuring apparatus (microscopic FT-IR measuring apparatus).

  The light source controller 141 is connected to the power source 142 and controls the output voltage of the power source 142 to adjust the intensity of the probe light emitted from the light source 143.

  The power source 142 supplies a voltage to the light source 143.

  The light source 143 emits probe light toward a predetermined position on the metal structure 110. The probe light emitted by the light source 143 is, for example, infrared probe light.

  The lens 144 narrows down the probe light emitted from the light source 143.

  The interferometer 145 forms an interference wave from the probe light narrowed down by the lens 144.

  The detection unit 146 detects information on the interference wave that has passed through the metal structure 110.

  The absorption spectrum calculation unit 147 converts the information of the transmission interference wave detected by the detection unit 146 into an electric signal and performs Fourier transform, and calculates an absorption spectrum of localized plasmon resonance on the metal structure 110.

  The computer unit 150 includes a control unit 152, a shift amount calculation unit 154, and an analysis unit 156.

  The control unit 152 comprehensively controls the substrate transfer device 120, the stage driving device 132, and the absorption spectrum measuring device 140.

  The shift amount calculation unit 154 complements the absorption spectrum of the localized plasmon resonance (hereinafter referred to as “reference spectrum”) measured in a state where one end of the complementary substance is fixed on the metal structure 110 and the metal structure 110. Compared to the absorption spectrum of the localized plasmon resonance (hereinafter referred to as “sensing spectrum”) measured with the target substance fixed and the sample containing the detection target substance introduced, the shift amount (Δλ) of the absorption spectrum is calculated. calculate. Specifically, for example, the shift amount (Δλ) is the difference between the maximum wavelength of the reference spectrum and the maximum wavelength of the sensing spectrum. Further, the reference spectrum is measured in advance and stored in a storage device (not shown), for example.

  The analysis unit 156 analyzes the detection target substance included in the sample using the shift amount (Δλ) calculated by the shift amount calculation unit 154. Specifically, for example, the analysis unit 156 uses the calibration curve indicating the relationship between the amount of shift of the absorption spectrum (Δλ) and the amount of the detection target substance to determine the shift amount (Δλ) of the absorption spectrum of the detection target substance. The amount of the detection target substance is analyzed by converting it into the amount. The calibration curve is created in advance and stored in a storage device (not shown).

  In addition, the relationship between the shift amount (Δλ) of the absorption spectrum and the amount of the detection target substance varies depending on the detection target substance and the complementary substance, but is generally known to be proportional. Therefore, it is possible to create a calibration curve using a shift amount (Δλ) that is a unique physical quantity of a state to be detected (a conjugate of a substance to be detected and a complementary substance), thereby realizing quantitative sensing.

  The input unit 160 has a function as an interface of the computer unit 150 and converts an operation signal from the user into a data format that can be processed by the computer unit 150.

  The output unit 170 is, for example, a display and a printer, and outputs information indicating the detection target substance contained in the sample analyzed by the analysis unit 156.

  Next, the manufacturing method of the metal structure 110 is demonstrated using FIG. 5 and FIG. FIG. 5 is a flowchart for explaining the manufacturing procedure of the metal structure. Moreover, FIG. 6 is sectional drawing according to process for demonstrating the manufacturing procedure of a metal structure.

  As described above, the size, shape and direction of the metal nanostructures constituting the metal structure 110, and the distance between the metal nanostructures are aligned, and are arranged in a precise and regular manner. The shift amount of the absorption spectrum of plasmon resonance can be increased. As a result, sensing accuracy is improved.

  Here, the substrate on which the metal structure is formed is preferably a solid material that does not absorb light from the visible region to the near infrared region in order to operate as a light-responsive device. Of these, quartz, sapphire and the like are preferable.

  First, in step S1000, the solid transparent substrate 112 (for example, a glass substrate) is cleaned and dried (see FIG. 6A). If the surface of the solid transparent substrate 112 is not sufficiently cleaned, metal nanorods (blocks), which are metal nanostructures formed on the solid transparent substrate 112 in a later step, may be peeled off from the solid transparent substrate 112. The solid transparent substrate 112 is sufficiently cleaned and dried.

  In step S1100, a positive electron lithography resist solution is spin-coated on the surface of the solid transparent substrate 112 cleaned in step S1000, and then baked (heated) to remove the resist solution. A thin film 114 is formed over the solid transparent substrate 112 (see FIG. 6B).

  At this time, in order to realize miniaturization of metal nanorods (blocks) formed in a later step, the thickness of the resist thin film 114 formed on the solid transparent substrate 112 is preferably less than or equal to micrometers. Specifically, for example, it is preferably 200 nm or less. The present inventor has found that in order to form such a thin resist film 114, a resist solution obtained by diluting a commercially available resist with a dedicated solvent to about 2 times may be used for spin coating.

  Here, the reason why the film pressure of the resist thin film 114 is set to 200 nm or less in order to realize miniaturization of the metal nanorod (block) is as follows. If the resist film pressure is larger than 200 nm, the entire thick resist film must be exposed with an electron beam when drawing exposure is performed with an electron beam in a later step, and the acceleration voltage of the electron beam needs to be extremely increased. Occurs. In general, the spatial resolution of drawing can be improved as the acceleration voltage of the electron beam increases. However, with such an extremely high acceleration voltage, the spatial resolution of the drawing is rather lowered. Therefore, it is not necessary to extremely increase the acceleration voltage of the electron beam in order to achieve the spatial resolution for accurately drawing the metal nanostructure of the size assumed by the present invention, and the acceleration voltage required in this case Then, a film pressure of 200 nm or less is appropriate.

  In step S1200, a predetermined pattern is drawn on the resist thin film 114 formed in step S1100 by, for example, an electron beam exposure apparatus (not shown), developed, rinsed, and dried (see FIG. 6C). Here, the predetermined pattern is obtained by tracing an integrated layout of a desired metal nanorod (block) array.

At this time, in order to achieve the formation of a predetermined metal nanorod (block) in a later step (both the length in the major axis direction and the minor axis direction is 100 nm or less), the exposure condition of this electron beam exposure step is Optimization becomes extremely important. As a result of repeated detailed experiments, the present inventor has found the following optimization conditions. That is, as an optimization condition for exposure, it is preferable to increase the acceleration voltage of the electron beam and to significantly reduce the exposure dose rate. Specifically, for example, the acceleration voltage of the electron beam is preferably set to 100 kV to 200 kV, and the exposure dose rate is preferably set to ² μC / cm ² or less. By setting such exposure conditions, a fine metal structure can be formed. What should be noted is the condition of a significantly low dose rate. For example, as a guideline, the dose rate of 1 μC / cm ² is about 1/100 of the dose rate recommended for commercially available resists.

  Here, the reason why the acceleration voltage of the electron beam is increased and the exposure dose rate is significantly reduced as the optimization condition for exposure is as follows. As described above, the spatial resolution of processing (drawing) is improved by increasing the acceleration voltage of the electron beam. This is because if the acceleration voltage of the electron beam is increased, the speed of the electron beam is increased and the de Broglie wavelength of the electrons is shortened. On the other hand, increasing the exposure dose rate corresponds to increasing the exposure time. If the exposure time is lengthened, the vibration of the sample itself during exposure (for example, air conditioning noise in the laboratory or extremely fine vibration noise of the apparatus itself) cannot be ignored, and the “blurring” of the processed shape occurs, which causes processing. The resolution will be reduced. Therefore, it is appropriate to increase the acceleration voltage of the electron beam and significantly reduce the exposure dose rate as the optimization conditions for exposure.

  In this manufacturing method, the development time is also an important parameter. Corresponding to significantly reducing the exposure dose rate, the development time is preferably longer than the standard time. Specifically, for example, it is preferably about 30 minutes.

  In step S1300, chromium and gold are sequentially formed by sputtering on the solid transparent substrate 112 processed in step S1200 to form the metal film 116 (see FIG. 6D). Note that platinum or silver may be used as a metal to be formed over chromium.

  Here, the chromium layer is formed in order to enhance the adhesion between the solid transparent substrate 112 and gold, and the film thickness is preferably about 2 nm. Gold is a material that exhibits a plasmon resonance response, and the thickness of the gold layer is preferably 10 nm to 100 nm. This film thickness corresponds to the thickness of the metal nanorod (block) when the metal structure 110 is completed.

  In step S1400, as a final step of forming the metal nanorod (block) array, the metal nanorod (block) array 118 is removed by removing (peeling) the excess resist material from the solid transparent substrate 112 processed in step S1300. Is completed (see FIG. 6E). The resist removal in this step is performed, for example, by penetrating the solid transparent substrate 112 processed in step S1300 into a chemical solution called a registry mover and ultrasonically cleaning (lift-off).

  At this time, even if ultrasonic cleaning is performed at room temperature (room temperature), excess resist cannot be removed. The present inventor has found that excess resist can be completely removed by performing ultrasonic cleaning while heating the solid transparent substrate 112 and the registry mover to 65 ° C. to 70 ° C. That is, the present inventor has found that it is necessary to perform heating in addition to ultrasonic cleaning in order to remove excess resist in the lift-off process. Here, the reason why the resist removal efficiency is improved by heating in addition to ultrasonic cleaning is that the resist material is easily dissolved by the solvent.

  In step S1500, the metal nanorod (block) array 118 completed in step S1400 is evaluated by an electron microscope and optical measurement. Specifically, the microstructure of the completed metal nanorod (block) array 118 is clarified by an electron microscope, and the plasmon resonance absorption response to the completed metal nanorod (block) array 118 is measured by measuring an absorption spectrum using an optical microscope. To evaluate. Furthermore, the efficiency of multiphoton absorption is also evaluated using a laser as an excitation light source.

  Summarizing the actual evaluation results in step S1500, structurally, according to the manufacturing method of the metal structure according to the present embodiment, the size of the manufactured metal nanorod (block) (major axis direction / minor axis direction). (Length, thickness) can be controlled very well on a nanometer scale, and the size variability can be suppressed to 5% or less, respectively. Moreover, in the produced metal nanorod (block) array, the distance between adjacent metal nanorods (blocks) can be reduced to 50 nm or less, and the variability of the distance can be suppressed to 5% or less. Furthermore, the major axis or minor axis of each metal nanorod (block) can be aligned along one axial direction. Further, the design (size, shape, directionality, interval) of the metal nanorods (blocks) can be arbitrarily controlled by patterning of electron beam exposure drawing.

  Functionally, the metal nanorod (block) array produced by the above manufacturing method has a plasmon absorption characteristic (absorption) that greatly depends on the size of the metal nanorod (block) and the distance between adjacent metal nanorods (block). (Wavelength position) function (wavelength selectivity of optical response) can be expressed.

  According to this manufacturing method, it is possible to produce a metal nanorod (block) array in which a large number of metal nanorods (blocks) are integrated by aligning the size, shape and orientation of the metal nanorods (blocks) and the distance between the metal nanorods (blocks).

  Next, a detection procedure using the sensing device 100 will be described with reference to FIG. FIG. 7 is a flowchart for explaining a detection procedure using the sensing device according to Embodiment 1 of the present invention.

  First, in step S2000, one end of a complementary substance is fixed to the surface of the metal structure 110. At this time, it is preferable that one end of the complementary substance is chemically adsorbed and fixed to the surface of the metal structure 110, and more preferably, it is firmly fixed using a covalent bond. For example, if the metal structure 110 is gold, a thiol group (—SH) is bonded to one end of the complementary substance, and a gold atom-sulfur atom generated by a chemical reaction between the surface of the metal structure 110 and the thiol group. One end of the complementary substance is fixed to the surface of the metal structure 110 by the covalent bond. At this time, due to the intermolecular interaction of the complementary substance and the interaction between the complementary substance and the metal structure 110, the complementary substance is closely fixed by the formation of an assembly called self-assembly. That is, it is preferable to fix the densely by inducing a covalent bond of a gold atom-sulfur atom on the surface of the metal structure 110 to fix a complementary substance and further inducing self-organization.

  In step S2100, a sample containing the detection target substance is introduced onto the metal structure 110 on which the complementary substance is fixed. The metal structure 110 into which the sample is introduced is loaded into the substrate transfer device 120.

  In step S <b> 2200, the metal structure 110 is placed on the stage 130. Specifically, the substrate transfer device 120 performs placement by transferring the loaded metal structure 110 onto the stage 130 according to an instruction from the control unit 152. Further, as described above, the substrate transfer apparatus 120 can simultaneously load a plurality of metal structures 110 and transfer them onto the stage 130 in order to realize multi-sample multi-item sensing.

  In step S <b> 2300, probe light is emitted from the light source 143 toward a predetermined position on the metal structure 110. The probe light emitted from the light source 143 is narrowed down by the lens 144 and becomes an interference wave via the interferometer 145. At this time, it is preferable that the polarization characteristic of the probe light emitted from the light source 143 is linearly polarized light. In other words, it is preferable to adjust the polarization direction of the probe light so as to be parallel to the major axis direction of the metal nanostructure.

  In step S <b> 2400, the detection unit 146 detects information on interference waves that have passed through the metal structure 110.

  In step S2500, the absorption spectrum calculation unit 147 converts the information of the transmission interference wave detected by the detection unit 146 into an electrical signal and performs Fourier transform, thereby calculating the spectrum of localized plasmon resonance on the metal structure 110. To do.

  In step S2600, the shift amount calculation unit 154 compares the reference spectrum and the sensing spectrum to calculate the shift amount (Δλ) of the absorption spectrum. As described above, the shift amount (Δλ) is the difference between the maximum wavelength of the reference spectrum and the maximum wavelength of the sensing spectrum. Further, the reference spectrum is measured in advance and stored in a storage device (not shown), for example.

  In step S2700, the analysis unit 156 analyzes the amount of the detection target substance included in the sample using the shift amount (Δλ) calculated by the shift amount calculation unit 154. Specifically, for example, as described above, by using the calibration curve indicating the relationship between the shift amount (Δλ) and the amount of the detection target substance, the shift amount (Δλ) is converted into the amount of the detection target substance. , Analyze the amount of detection target substance. The calibration curve is created in advance and stored in a storage device (not shown).

  Here, as described above, the relationship between the shift amount (Δλ) and the amount of the detection target substance varies depending on the detection target substance and the complementary substance, but is generally known to be proportional. Therefore, it is possible to create a calibration curve using the shift amount (Δλ), which is a specific physical quantity of the state to be detected, and realize quantitative sensing. Further, the shift amount (Δλ) increases as the amount of the detection target substance increases.

  Here, the reason why the shift amount (Δλ) increases as the amount of the detection target substance increases is as follows. When the detection target substance and the complementary substance are combined, the dielectric constant (refractive index) in the vicinity of these combined substances is changed, and the absorption spectrum of the localized plasmon resonance is shifted in accordance with the change in the dielectric constant. At this time, the larger the amount of the detection target substance, the larger the change in the dielectric constant, so the shift amount (Δλ) increases. Note that the relationship between the change in dielectric constant and the shift amount (Δλ) is also proportional.

  In the present embodiment, the detection target substance and the complementary substance are the antigen in the antigen-antibody reaction and the sample DNA and the probe DNA in the hybridization of the antibody or DNA, but the present invention is not limited to this. For example, a so-called host-guest compound can be applied to the present invention as a detection target substance and a complementary substance. For example, a metal ion (guest) can be detected by immobilizing a crown ether compound having excellent ability to recognize each metal ion to the metal structure 110 as a probe (host). In addition, by fixing cyclodextrins as probes (hosts) to the metal structure 110, a wide variety of organic molecule groups can be detected.

  As described above, according to the present embodiment, sensing is performed using the absorption spectrum of localized plasmon resonance, which is an intrinsic physical quantity of the state to be detected, and thus the detection target substance is detected with high accuracy and quantitatively. be able to.

(Embodiment 2)
The present inventor further binds the metal nanoparticles to the other end of the probe DNA, one end of which is fixed to the metal structure, when the detection target substance is the probe DNA and the complementary substance is the sample DNA. The inventors have found that upon hybridization, the electromagnetic interaction between the metal structure, the composite (double-stranded) and the metal nanoparticles is increased, and a further significant change in dielectric constant is induced. With this knowledge, the shift amount (Δλ) of the absorption spectrum of localized plasmon resonance under the same condition can be further increased.

  Sensing device 200 according to the present embodiment further includes metal nanoparticles 210 in sensing device 100 according to the first embodiment. In the present embodiment, it is assumed that the complementary substance is the probe DNA 106 and the detection target substance is the sample DNA 108.

  The metal nanoparticle 210 is a metal (for example, gold, platinum, silver, etc.) having a nano-order particle size, and is bonded to the other end of the probe DNA 106 having one end fixed to the metal structure 110 (FIG. 8 ( A)). The metal nanoparticles 210 and the probe DNA 106 are preferably chemically bonded.

  When the probe DNA 106 and the sample DNA 108 are hybridized, the DNA chain length after the binding is shortened, so that the distance between the surface of the metal structure 110 and the metal nanoparticles 210 is shortened, and the metal structure 110 and the complex (double strand). In addition, since the electromagnetic interaction between the metal nanoparticles 210 and the metal nanoparticles 210 is increased, a more remarkable change in dielectric constant is induced than in the first embodiment (see FIG. 8B). Therefore, the shift amount (Δλ) of the absorption spectrum of localized plasmon resonance under the same condition can be further increased.

  Thus, according to the present embodiment, the metal nanoparticle is bound to the other end of the probe DNA, one end of which is fixed to the metal structure. Therefore, during the hybridization, the metal structure and the complex (double strand) Electromagnetic interaction between the metal nanoparticles and the metal nanoparticles is increased, and a remarkable change in dielectric constant is induced. The shift amount (Δλ) of the absorption spectrum of the localized plasmon resonance under the same condition can be further increased. As a result, the detection target substance can be detected with higher accuracy than in the first embodiment.

  The inventor conducted detailed experiments in order to demonstrate the effects of the present invention. Hereinafter, more specific embodiments (examples) of the present invention will be described. In addition, this invention is limited to a following example and is not interpreted.

  In Example 1, a metal structure is produced by producing metal nanoblocks having different aspect ratios by electron beam lithography / lift-off on a glass substrate (Matsunami Glass: 24 mm × 24 mm), and a detection target substance The sensing function of was tested.

Specifically, first, the glass substrate was ultrasonically cleaned using acetone, methanol, and ultrapure water in this order. Next, a positive electron resist (ZEP-520A manufactured by Nippon Zeon Co., Ltd., diluted twice with a dedicated thinner) is spin-coated on a glass substrate (initial: 1000 rpm for 10 sec, main: 4000 rpm for 90 sec), Pre-baking (3 minutes at 180 ° C.) was performed on a hot plate. Next, after drawing a predetermined pattern at a dose rate of 1.2 μC / cm ² using an electron beam exposure apparatus with an acceleration voltage of 100 kV, development (30 minutes), rinsing and drying were performed. Next, chromium and gold were sequentially formed on the glass substrate by sputtering to form a metal film. Next, it was infiltrated into a registry mover (dimethylformamide) solution heated to 65 ° C. to 70 ° C. and subjected to ultrasonic cleaning (5 minutes).

  As for the size of the metal structure manufactured as described above, the length in the major axis direction, the length in the minor axis direction, and the thickness could be controlled on a scale of the order of several tens nm to several hundreds nm, respectively. .

  Next, the detection function of the sensing device using the produced metal structure was tested.

  Specifically, an organic solvent having a different refractive index (about 1.0 to 1.7) is introduced onto a metal structure composed of metal nanoblocks having a different aspect ratio (1 to 9), and localized. The shift amount (Δλ) of the absorption spectrum of plasmon resonance was measured. For the measurement, a Fourier transform type micro infrared spectrophotometer was used.

  FIG. 9A is a diagram showing the relationship between the refractive index of an organic solvent and the shift amount (Δλ) of the absorption spectrum of localized plasmon resonance. In addition, a metal structure composed of five types of metal nanoblocks (major axis length of 100 nm or more and minor axis length of 100 nm or less) having aspect ratios of 1, 3, 5, 7, and 9, respectively. The experiment was conducted using the body. FIG. 9B is a diagram showing the relationship between the aspect ratio of the metal nanoblock and the shift amount Δλ / refractive index n.

  From FIG. 9 (A), the refractive index of the organic solvent and the shift amount (Δλ) of the absorption spectrum of the localized plasmon resonance are proportional to each other regardless of the metal structure composed of the metal nanoblock of any aspect ratio. It can be seen that For example, in the measurement of a metal structure composed of gold nanoblocks with an aspect ratio of 9, a refractive index change Δn = 0.01 corresponds to a shift amount (Δλ) = 4 (nm). FIG. 9B shows that the higher the aspect ratio of the gold nanoblock is, the larger the shift amount (Δλ) is, and the highly accurate detection is realized.

  As described above, when a substance to be detected and a complementary substance are bound to each other, the refractive index in the vicinity of these bound substances is changed, and the absorption spectrum of the localized plasmon resonance is shifted with the change in the refractive index. In this embodiment, the change in the refractive index due to the binding between the detection target substance and the complementary substance corresponds to the difference in the refractive index of the organic solvent. Therefore, the effect of the present invention can be demonstrated by this example in which an experiment was performed using an organic solvent.

  Next, one end of the probe DNA was fixed to the metal structure prepared as described above, and metal nanoparticles were bound to the other end of the probe DNA to test the sensing function of the detection target substance. In addition, gold was used for the metal nanoparticles.

  Specifically, first, the metal structure was immersed in a concentrated nitric acid solution (for 3 minutes) and washed in a cleaning solution (sodium dodecyl sulfate solution) heated to 50 ° C. with stirring (5 minutes × 3 times). Next, a synthetic nucleotide (40 bases) in which a thiol group is bonded to the 3 ′ end and biotin is bonded to the 5 ′ end group is immobilized on the metal structure by thiol bonding (37 ° C., 10 hours) to probe DNA. It was. Next, by dropping a mercaptoacetic acid solution onto the metal structure to negatively charge the surface of the metal structure, and adding and immersing the dispersion solution (4 hours), gold nanoparticles having a surface coated with streptavidin ( 10 nm in diameter) and the 5 ′ end of the probe DNA were bound by biotin-streptavidin interaction.

  In this way, one end of the probe DNA was fixed on the metal structure, and the other end of the probe DNA was bonded to the gold nanoparticles. At this time, the surface of the metal nanoblock on the metal structure was covered with a monomolecular film of single-stranded probe DNA at a coverage of about 30%.

  In addition, the produced sample was observed with an electron microscope. FIG. 10A is an electron showing a sample in which one end of the probe DNA is fixed on a metal structure (configured by a gold nanoblock having an aspect ratio = 1 (100 nm × 100 nm)) and the other end is bonded to a gold nanoparticle. It is a micrograph. FIG. 10 (B) is an electron showing a sample in which one end of the probe DNA is fixed on a metal structure (configured by a gold nanoblock having an aspect ratio = 6 (50 nm × 300 nm)) and the other end is bonded to a gold nanoparticle. It is a micrograph.

  10A and 10B, a large number of gold nanoparticles (diameter 10 nm) bound to the other end of the probe DNA were observed regardless of the aspect ratio of the gold nanoblock.

  Next, the detection function of the sensing device using the produced metal structure was tested.

  Specifically, a solution containing a sample DNA having a complementary relationship with the probe DNA is introduced into the prepared metal structure to induce hybridization, and a Fourier transform type micro-infrared spectrophotometer is used on the sample. The absorption spectrum of localized plasmon resonance at a predetermined position was measured. Further, in order to demonstrate the superiority of bonding gold nanoparticles to the other end of the probe DNA, a similar experiment was also performed on a metal structure that does not bind gold nanoparticles to the probe DNA. For the measurement, an experiment was performed using a metal structure composed of three kinds of metal nanoblocks having aspect ratios of 1, 5, and 9, respectively.

  FIG. 11A is a diagram showing an absorption spectrum of localized plasmon resonance at a predetermined position on a metal structure that does not bind gold nanoparticles to the other end of the probe DNA. FIG. 11B is a diagram showing an absorption spectrum of localized plasmon resonance at a predetermined position on a metal structure in which gold nanoparticles are bonded to the other end of the probe DNA. FIG. 11C is a diagram showing the relationship between the aspect ratio of the gold nanoblocks in FIG. 11A and FIG. 11B and the shift amount (Δλ) of the absorption spectrum of the localized plasmon resonance. Note that FIGS. 11A to 11B are obtained by using a metal structure including metal nanoblocks having an aspect ratio = 9.

  In FIG. 11A, spectrum P is an absorption spectrum of localized plasmon resonance when neither probe DNA nor sample DNA is immobilized on the metal structure, and spectrum Q is a probe on the metal structure. This is an absorption spectrum of localized plasmon resonance when only DNA is immobilized, and spectrum R is an absorption spectrum of localized plasmon resonance when a sample containing sample DNA is introduced onto a metal structure on which probe DNA is immobilized. is there. Therefore, the shift amount (Δλ) in FIG. 11A is the difference between the maximum wavelength of the spectrum Q and the maximum wavelength of the spectrum R.

  In FIG. 11B, spectrum P ′ is an absorption spectrum of localized plasmon resonance when neither probe DNA nor sample DNA is immobilized on the metal structure, and spectrum Q ′ is on the metal structure. Is the absorption spectrum of localized plasmon resonance when only the probe DNA is immobilized, and the spectrum R ′ is the spectrum of localized plasmon resonance when the sample containing the sample DNA is introduced onto the metal structure on which the probe DNA is immobilized. It is an absorption spectrum. Therefore, the shift amount (Δλ) in FIG. 11B is the difference between the maximum wavelength of the spectrum Q ′ and the maximum wavelength of the spectrum R ′.

  From FIG. 11 (A) and FIG. 11 (B), it was found that the absorption spectrum of plasmon resonance was shifted by hybridization. It was also found that the shift amount (Δλ) of the absorption spectrum of localized plasmon resonance under the same conditions can be increased by bonding gold nanoparticles to the other end of the probe DNA. For example, the maximum shift amount (Δλ) in FIG. 11B was 40 nm. Furthermore, FIG. 11C shows that the shift amount (Δλ) of the absorption spectrum of localized plasmon resonance under the same conditions can be increased as the aspect ratio of the gold nanoblock is higher (aspect ratio = 9).

  At this time, in FIG. 11A, the refractive index change Δn = 0.025 corresponds to the shift amount (Δλ) = 10 (nm). In addition, since the refractive index of the sample DNA itself was 1.46 and the surface coverage of the gold nanoblock was 30%, the proportion of the hybridized DNA was estimated to be 18%.

  Since the sensing method and the sensing device according to the present invention can detect a detection target substance with high accuracy and quantity without using fluorescence sensing or light reflection, a sensing method used for medical diagnosis, genetic analysis, and environmental analysis And it is useful as a sensing device.

The figure which shows the whole structure of the sensing apparatus which concerns on Embodiment 1 of this invention. The block diagram which shows the structure of the sensing apparatus shown in FIG. (A) The figure which shows the state by which the end of the antibody was fixed on the immunoassay chip, (B) The figure which shows the state in which the antibody fixed on the immunoassay chip and the introduced antigen selectively formed a complex. (A) A diagram showing a state in which one end of the probe DNA is fixed on the DNA chip, (B) a diagram showing a state in which the probe DNA fixed on the DNA chip and the introduced sample DNA form a complex. Flow chart for explaining the manufacturing procedure of the metal structure Sectional drawing according to process for demonstrating the manufacturing procedure of a metal structure The flowchart for demonstrating the detection procedure using the sensing apparatus shown in FIG. 1 and FIG. (A) The figure which shows the state which the metal nanoparticle couple | bonded with the other end of probe DNA, (B) The figure which shows the state which probe DNA of (A) hybridized with sample DNA (A) The figure which shows the relationship between the refractive index of an organic solvent, and the shift amount ((DELTA) (lambda)) of the absorption spectrum of a local plasmon resonance, (B) The relationship between the aspect-ratio of metal nanoblock and shift amount (DELTA) (lambda) / refractive index n. Illustration (A) An electron micrograph showing a sample in which one end of the probe DNA is fixed on a metal structure (configured by gold nanoblocks having an aspect ratio = 1 (100 nm × 100 nm)) and the other end is bonded to gold nanoparticles; B) Electron micrograph showing a sample in which one end of the probe DNA is fixed on a metal structure (configured with gold nanoblocks having an aspect ratio of 6 (50 nm × 300 nm)) and the other end is bonded to gold nanoparticles. (A) A diagram showing an absorption spectrum of localized plasmon resonance at a predetermined position on a metal structure (configured by gold nanoblocks having an aspect ratio of 9) that does not bind gold nanoparticles to the other end of the probe DNA. FIG. 11C shows an absorption spectrum of localized plasmon resonance at a predetermined position on a metal structure (configured by gold nanoblocks having an aspect ratio = 9) in which gold nanoparticles are bonded to the other end of the probe DNA. The figure which shows the relationship between the aspect-ratio of a gold nanoblock in FIG. 11 (A) and FIG. 11 (B), and the shift amount ((DELTA) (lambda)) of the absorption spectrum of a localized plasmon resonance.

Explanation of symbols

100, 200 Sensing device 102 Antibody 104 Antigen 106 Probe DNA
108 Sample DNA
DESCRIPTION OF SYMBOLS 110 Metal structure 112 Solid transparent substrate 114 Resist thin film 116 Metal film 118 Metal nanorod (block) array 120 Substrate transfer device 130 Stage 132 Stage drive device 140 Absorption spectrum measuring device 141 Light source controller 142 Power source 143 Light source 144 Lens 145 Interferometer 146 Detection Unit 147 absorption spectrum calculation unit 150 computer unit 152 control unit 154 shift amount calculation unit 156 analysis unit 160 input unit 170 output unit 210 metal nanoparticles

Claims (5)

Preparing a metal structure in which a plurality of metal nanostructures exhibiting localized plasmon resonance, on which probe DNA capable of hybridizing to DNA to be detected is immobilized, are arranged on a transparent substrate;
Providing a sample solution to a region where the plurality of metal nanostructures are disposed on the surface of the metal structure;
Irradiating the metal structure provided with the sample liquid with probe light and detecting transmitted light;
Detecting the DNA to be detected contained in the sample liquid from the shift amount of the maximum wavelength of the absorption spectrum of the transmitted light, and a method for detecting DNA comprising:
The plurality of metal nanostructures have the same shape with an aspect ratio of 5 or more, and are regularly arranged on the transparent substrate at regular intervals so that their long axes are parallel to each other,
The probe DNA has one end fixed to the surface of the metal nanostructure, and the other end is bonded with metal nanoparticles.
DNA detection method. The probe light is linearly polarized light,
The polarization direction of the probe light is parallel to the major axis direction of the plurality of metal nanostructures,
The method for detecting DNA according to claim 1. A transparent substrate;
A plurality of metal nanostructures disposed on the transparent substrate and exhibiting localized plasmon resonance;
A metal structure for DNA detection, comprising probe DNA immobilized on the surface of the metal nanostructure and capable of hybridizing to DNA to be detected;
The plurality of metal nanostructures have the same shape with an aspect ratio of 5 or more, and are regularly arranged on the transparent substrate at regular intervals so that their long axes are parallel to each other,
The probe DNA has one end fixed to the surface of the metal nanostructure, and the other end is bonded with metal nanoparticles.
Metal structure for DNA detection. The metal structure according to claim 3, wherein a sample liquid is provided on the surface;
A light irradiation unit for irradiating the metal structure with probe light; and
A transmitted light detector that detects transmitted light transmitted through the metal structure;
From the amount of shift of the maximum wavelength of the absorption spectrum of the transmitted light, an analysis unit for detecting the DNA to be detected contained in the sample solution;
An apparatus for detecting DNA. The probe light is linearly polarized light,
The polarization direction of the probe light is parallel to the major axis direction of the plurality of metal nanostructures,
The DNA detection apparatus according to claim 4.