JP2009014491A - Target material detection element and target material detection device - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a target material detection element capable of intensifying sufficiently a Raman scattering signal of a target material. <P>SOLUTION: This target material detection element has a support 101; a metal nanodot 102 existing on the surface of the support; and capturing molecules 103 existing on the surface of the metal nanodot 102, for capturing a target material in a specimen. The metal nanodot 102 has n-pieces of spectrum peaks of plasmon resonance, and each peak wavelength to the n-th large peak among peaks carried by a Raman band of the target material in the specimen and each peak wavelength of the n-pieces of peaks carried by the metal nanodot 102 exist nearby in association with each other. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  The present invention relates to a target substance detection element for detecting a target substance in a specimen with high sensitivity.

  In the medical field, a technique for monitoring a marker that causes a disease is expected as a next-generation technique, and is also expected to greatly contribute to improvement of medical quality.

  There are multiple markers in the blood for specific diseases such as cancer, hepatitis, diabetes, or osteoporosis. When affected, the concentration of, for example, a specific protein that becomes a marker from normal time changes (increases or decreases). By monitoring the concentration of these proteins from normal time, a serious disease can be detected early. In addition, when performing prognosis after tumor removal, tumor metastasis or recurrence can be detected early by monitoring the concentration of the protein serving as a marker.

  Examples of methods for analyzing raw and unpurified proteins include fluorescence immunization, plasmon resonance, and optical interference.

  Both methods utilize biological ligand-analyte interactions. In the ligand-analyte interaction, a ligand that specifically binds to the analyte to be analyzed in the specimen is immobilized on the sensor surface, and the analyte is selected with high selectivity. As a result, contaminants in the sample are removed, and the analyte to be analyzed can bind to the ligand on the sensor surface with high efficiency.

  On the other hand, in addition to the above-described method, surface-enhanced Raman spectroscopy (hereinafter referred to as “SERS (Surface Enhanced Raman Spectroscopy) method”) has attracted attention in recent years.

In the SERS method, a metal nanostructure such as a metal thin film or a metal colloid is fixed on the sensor surface, and when the sensor surface is irradiated with excitation laser light, an evanescent electric field is induced around the metal nanostructure. When the evanescent electric field is induced, the Raman scattering cross section is enhanced to about 10 ³ to 10 ¹⁴ times, and the Raman scattering signal of the molecules and materials adsorbed on the metal nanostructure is enhanced.

  Therefore, by detecting the intensity of the Raman scattering signal from the Raman active molecule labeled on the analyte, the analyte as the target substance is identified, and the concentration of the analyte is also measured. Further, as additional information, it is possible to detect information on bond bonds generated by the bond between the ligand and the analyte and other information related to the bond between the ligand and the analyte.

  Here, the characteristics of the SERS method are summarized as follows.

(1) Very large electric field increase In the very vicinity of the metal nanostructure, the Raman scattering cross section is enhanced to about 10 ³ to 10 ¹⁴ times. Therefore, when the SERS method is applied to an immunoassay, the detection sensitivity is high and the detection limit can be greatly reduced.

(2) Reduction of autofluorescence Biofluorescence derived from living organisms appears in the same wavelength band as the Raman scattering signal. In the SERS method, since the metal nanostructure is used for the sensor surface, the autofluorescence is absorbed by the metal nanostructure, and the background can be reduced.

(3) No need for B / F separation In the SERS method, the Raman scattering signal from the molecule immobilized around the metal nanostructure or the molecule adsorbed on the immobilized molecule is specifically enhanced. Therefore, a Raman scattering signal of a desired molecule can be obtained.

(4) Superiority of Raman active molecule In the SERS method, it is possible to employ a technique of labeling with a Raman active molecule that is stable and has a low possibility of fading compared to a fluorescent molecule or the like. Multiple types of Raman-active molecules can be used simultaneously.

  Taking advantage of these features, attempts have been made to analyze organic molecules and immunoassay using the SERS method.

  For example, the SERS method described in Patent Document 1 discloses a technique for enhancing a Raman scattering signal by immobilizing a metal nanostructure on a sensor surface in a single particle film state. In Patent Document 1, the metal nanostructures are randomly distributed with respect to the deflection direction of the incident electric field, and the Raman scattering signal is enhanced by the generated plasmon peak. Plasmon means that free electrons in a metal collectively vibrate by light, and the wavelengths absorbed by the metal are different. The plasmon peak is a wavelength that absorbs light most strongly when plasmon occurs.

  In addition, the SERS method described in Patent Document 2 discloses a technique for enhancing the Raman scattering signal by utilizing the fact that the electric field becomes strong in the region of the edge portion such as a nano triangular prism.

  The SERS method described in Non-Patent Document 1 discloses a technique for detecting a protein on the surface of cancer cells. The metal colloid is labeled with MGITC (Malachite green isothiocyanate), which is a kind of Raman active molecule, and an antibody is bound to the metal colloid. The method of Non-Patent Document 1 is excellent in portability, such as detecting protein on the cell surface.

In addition, the SERS method described in Non-Patent Document 2 discloses a technique for measuring each Raman band of Rhodamine 123. Non-Patent Document 2 changes the position of the plasmon peak by changing the aggregation state of the silver nanoparticles. A Raman band is a unique band that appears in an atomic bond or atomic group.
Japanese Patent Laying-Open No. 2005-233637 JP 2005-219184 A Dominic O. Ansari, Douglas A. Stuart, and Shuming Nie (2005), Proceedings of SPIE-Volume 5699 pp. 82-90 Kenichi Yoshida, Tammu Ito, Yasuo Yoshikawa, Yukihiro Ozaki (2005) General Meeting of Molecular Structures Proceedings 3P131

  However, in the SERS method for organic molecule analysis and immunoassay as described above, the wavelength position of the plasmon peak and the Raman band of the target substance do not always match. In that case, there is a problem that the Raman scattering signal is not sufficiently enhanced.

  An object of the present invention is to provide a target substance detection element that can sufficiently enhance the Raman scattering signal of the target substance.

The present invention
A target substance detection element for detecting a target substance in a specimen,
A support;
Metal nanodods present on the surface of the support;
A capture molecule that is present on the surface of the metal nanodot and captures a target substance in the specimen;
The metal nanodot has n plasmon spectrum peaks;
The target substance characterized in that the peak wavelength of the n-th largest peak among the peaks of the Raman band of the target substance and the peak wavelengths of the n peaks of the metal nanodots are respectively present in the vicinity. It is a detection element.

  The metal nanodots are preferably composed of a plurality of types of metal nanodots having different absorption wavelengths.

  It is preferable that the metal nanodot has anisotropy.

  The metal nanodots are preferably composed of a plurality of types of rod groups having different shapes, and the angle θ between the main axis direction of the rod and the deflection direction of the excitation light of the rod is preferably as follows.

(I is the type of rod group, X (i) is the length in the major axis direction of each type of rod, Y (i) is the length in the minor axis direction of each type of rod, and X1 is the wavelength of the plasmon peak. (The length in the major axis direction of the short rod, Y1 represents the length in the minor axis direction of the rod with the shortest plasmon peak wavelength)
Another aspect of the present invention is:
The target substance detection element;
A first light source that emits light for inducing an evanescent electric field in the vicinity of the metal nanodots of the target substance detection element;
A second light source for irradiating the metal nanodots with light for causing Raman scattering;
And a spectroscope for detecting the Raman scattered light enhanced by the metal nanodots.

  The wavelength of the light from the second light source is preferably set to a wavelength that does not excite the autofluorescence of impurities contained in the specimen.

  It becomes possible to detect the target substance in the specimen with high sensitivity.

  Embodiments for carrying out the present invention will be described in detail with reference to the drawings. With reference to FIG. 1, the substrate for element formation and the target substance detection element for target substance detection are demonstrated. This target substance detection element has a support 101, metal nanodots 102, and a trap 103.

  The element formation substrate is composed of a support 101 and metal nanodots 102, and the planar metal nanodots 102 are regularly arranged on the surface of the support 101.

  Further, the target substance detection element includes a substrate for element formation and a capture body 103 for the target substance, and the capture body 103 is fixed to the metal nanodots 102. The target substance detection kit for producing the target substance detection element has the element forming substrate and the capture body 103, and a reagent for fixing the capture body 103 to the metal nanodots 102 as necessary. Also equipped. For example, the reagent is a solution containing IgG (immunoglobulin G) or IgE (immunoglobulin E), and can be provided as an aqueous solution or various buffers.

  The metal nanodot 102 has a spectrum of plasmon resonance, and has n (n is a natural number) spectrum peaks. Here, the spectrum peak indicates the maximum value of the corrected spectrum obtained by fitting the spectrum.

  Further, the peak wavelength of the n plasmon resonance spectral peaks of the metal nanodots corresponds to the peak wavelength of the nth largest peak among the peaks of the Raman band of the target substance in the specimen. It exists in the vicinity.

  Here, the peak wavelength of the spectrum peak of plasmon resonance and the peak wavelength of the Raman band peak of the target substance are in the vicinity. The peak wavelength of the peak of the Raman band of the target substance is in the half-width wavelength region of the spectrum of plasmon resonance. Is assumed to exist. More preferably, the peak wavelength of the Raman band peak of the target substance exists in the wavelength range of 3/4 width.

  It should be noted that the peak wavelength of n plasmon resonance spectrum peaks and the peak wavelength of the n-th largest peak among the peaks of the Raman band of the target substance in the sample correspond to each other in the vicinity. It has the following meaning.

  That is, one of the n plasmon resonance spectral peaks and one of the nth largest peaks in the Raman band of the target substance are present in the vicinity, so that there are n plasmon resonances. There is a one-to-one correspondence between the peak and the peak of the nth Raman band.

  As long as it has such properties, the metal nanodots 102 may have any shape. Preferably, the structure is a structure having a plasmon resonance wavelength in the infrared band, and suppresses the autofluorescence of the sample when irradiated with excitation light in the infrared band, so that the Raman scattering signal is converted into S / N (signal to noise ratio Signal). / Noise) The shape can be measured well.

  Moreover, as a kind of metal nanodot, one type may be sufficient and it may be comprised from multiple types. Preferably, the metal nanodots are composed of a plurality of types and have different absorption wavelengths. According to this, the Raman scattering peak can be uniformly enhanced in a wide band.

  The capturing body 103 is not particularly limited as long as it can form a specific binding pair with the target substance and can be immobilized on the metal nanodot 102. Here, it is preferable to use an antibody or a nucleic acid.

  The target substance to be detected by the capturing body 103 can be classified into a non-biological substance and a biological substance. Non-biological substances are endocrine disruptors called environmental hormones. Endocrine disrupting substances include environmental pollutants, such as PCBs (Polychlorinated Biphenyls) and dioxins having different positions from the number of chlorine substitutions.

  Biological substances are nucleic acids, proteins, sugar chains, lipids, and complexes thereof. Biological materials include DNA (Deoxyribonucleic Acid), RNA (RiboNucleic Acid), aptamer, gene, chromosome, cell membrane, virus, antigen, antibody, lectin, hapten, hormone, receptor, enzyme, peptide, sphingosaccharide, sphingolipid It is. In addition, bacteria and cells themselves that produce biological substances can also be target substances.

  Next, the configuration of the target substance detection device will be described with reference to FIG. The target substance detection apparatus includes a support 101, metal nanodots 102, light sources 202 and 206, a filter 203, a condenser lens 204, a collimating lens 207, a dichroic mirror 208, an objective lens 209, a total reflection mirror 210, a lens 211, and an aperture 212. , Lens 213, aperture 214, notch filter 215, and spectrometer 218.

  The light source 202 irradiates the metal nanodots 102 on the support 101 with the excitation light 205 through the filter 203 and the condenser lens 204 to induce an evanescent electric field around the metal nanodots 102.

  The light source 206 irradiates the dichroic mirror 208 with a laser beam 216 for generating Raman scattering via a collimator lens 207. When the light emitted from the light source 206 is incident, the dichroic mirror 208 irradiates the metal nanodots 102 disposed on the support 101 via the objective lens 209, and generates elastic scattering or inelastic Raman scattering.

  The spectroscope 218 measures the spectrum of the wavelength based on the incident light when the Raman scattered light Raman-scattered by the metal nanodot 102 is incident through the optical systems 210 to 215. The spectrum is a combination of atomic bonds or atomic groups and their bands, and a peak appears at a specific wave number. The target substance can be identified from the peak wave number, and the concentration of the target substance can be measured based on the light intensity.

  Next, a method for producing a target substance detection element will be described with reference to FIG. First, in the target substance detection element, a Ti layer (not shown) is deposited as a bonding (binder) layer to a thickness of 5 nm on a quartz wafer support 401, and an Au layer 402 is continuously deposited to a thickness of 50 nm (FIG. 3A). )reference).

  Subsequently, a negative resist (Shipley Far East, SAL601) is spin-coated to provide a resist layer 403 (see FIG. 3B). After pre-baking, when the support 401 provided with the resist layer 403 is introduced into the chamber of the electron beam drawing apparatus, a desired pattern is drawn on the resist layer 403 (see FIG. 3C). Subsequently, post-exposure baking is performed to quench the support 401. Thereafter, the support 401 is developed with a developer, and the resist layer 403 is patterned (see FIG. 3D).

  Dry etching is performed using the patterned resist layer as a mask. Then, when the pattern is transferred to the Au layer 402, nanodots are obtained. Finally, when a process such as a remover is performed, the resist layer 403 on the nanodots is removed (see FIG. 3E).

  Through the above process, rectangular metal nanodots as shown in an SEM (Scanning Electron Microscope) image as shown in FIG. 4A are formed and arranged in parallel with the direction of deflection of the incident electric field. And a target substance detection element is produced by fixing the capture body of Anti-DNP (Anti-Dinitrophenyl) to the surface of metal nanodot.

  Hereinafter, the present invention will be described in more detail with reference to examples. Here, an example in which an immune response is measured using a Raman active molecule and an example in which an immune response is measured without using a Raman active molecule will be described. First, an example in which an immune reaction is measured using a Raman active molecule will be described in detail.

Example 1
With reference to FIG. 1, the structure of the target substance detection element in a present Example is demonstrated. The target substance detection element includes a support 101, metal nanodots 102, and a trap 103.

  As the support 101, a quartz wafer having a height of 525 μm is used.

  The metal nanodot 102 is a rectangular parallelepiped element having a long side of 200 nm, a short side of 50 nm, and a height of 200 nm, and the material of the metal nanodot is gold. The length of the long side and the length of the short side of the metal nanodot 102 are lengths at which the plasmon peak and the wavelength position of the Raman band of the target substance coincide.

  Further, the metal nanodots 102 are arranged on the surface of the support 101 perpendicular to the incident light so that the main axis is in the same direction as the incident light. According to this, since the plasmon resonance occurs most strongly, the Raman scattering signal is enhanced, and the Raman band of the target substance can be measured with high sensitivity.

  Further, the metal nanodots 102 have anisotropy for defining an angle with incident light. According to this, since the intensity of the Raman scattering signal is changed by changing the angle with the incident light, the Raman band of the target substance can be measured with the required intensity.

  The capturing body 103 is an antibody.

Next, the immunoassay in this example will be described. Anti-DNP is immobilized on the surface of the metal nanodots to produce a target substance detection element. A specimen solution containing 1.5 × 10 ⁵ M DNBA (2,4-Dinitrobenzonic Acid) is incubated on the prepared target substance detection element to perform a binding reaction for 2 hours. Thereafter, when the sample solution is replaced with a phosphate buffer, the complex of DNBA and Anti-DNBA shown in FIG. 5 is immobilized on the surface of the metal nanostructure.

  Next, a method for measuring the target substance will be described. The target substance detection apparatus shown in FIG. 2 measures the spectrum of the Raman scattering signal of the complex of antiDNP-DNBA.

  First, when the excitation light 205 is irradiated from the light source 202 to the metal nanodot 102, a strong evanescent electric field is induced around the metal nanodot.

  Subsequently, when the laser light near the absorption wavelength (near infrared band) of the metal nanodot 102 is irradiated from the light source 206 to the metal nanodot 102, Raman scattering occurs in the metal nanodot 102. The spectroscope 218 measures the spectrum of light that has been Raman-scattered by the metal nanodots 102. Note that the wavelength is set so as not to excite the autofluorescence of impurities contained in the specimen. According to this, it is possible to measure the Raman scattered light by suppressing the autofluorescence due to the impurity impurities contained in the sample solution to a level where there is no practical problem.

  FIG. 4B is a diagram obtained by measuring an absorption spectrum in the atmosphere of the metal nanostructure. 4B, the horizontal axis represents wavelength (nm) and the vertical axis represents absorbance (OD). The metal nanodot 102 which is a metal nanostructure is a structure having an absorption peak near 920 nm in the atmosphere. It is known from various experiments and examinations that the metal nanodot 102 has a characteristic of approximately 350 nm / index. The refractive index of the atmosphere is 1.0, and the refractive index when introduced into the sample solution is 1.4. Therefore, when the sample is actually introduced into the sample solution, the absorption peak wavelength of the metal nanodot 102 is calculated as 920 + 350 × (1.4−1.0) = 1060 nm.

FIG. 6 is a diagram showing a Raman scattering spectrum in this example. In FIG. 6, the horizontal axis is the spectrum shift (cm ⁻¹ ), and the vertical axis is the intensity. In Figure 6, 1549cm ^-1, stretching vibration of a benzene ring in the vicinity of 1525 cm ^-1, stretching vibration of NO2 in the vicinity of 1338cm ^-1, it can be confirmed stretching vibration near 839cm ^-1. In this case, excitation light, semiconductor laser: excitation is performed at 980 nm, and therefore, when described in terms of wavelength, there are peaks in the vicinity of 1155 nm, 1152 nm, 1128 nm, and 1067 nm.

When the sample solution is present on the surface of the nanostructure, the absorption spectrum of plasmon resonance has an absorption peak at about 1060 nm, so that the stretching vibration around 839 cm ⁻¹ is the best S / N.

  According to this example, the plasmon peak of the metal nanodot matches the wavelength position of the Raman band of the target substance, so that the Raman scattering signal of the target substance can be sufficiently enhanced.

(Example 2)
First, the configuration of the target substance detection element will be described with reference to FIG. 7A. The metal nanodot uses two rectangular parallelepiped elements having anisotropy for defining an angle with incident light, and the metal nanodot 1020 is an element having a long side of 200 nm, a short side of 50 nm, and a height of 20 nm. The metal nanodot 1021 is an element having a long side of 220 nm, a short side of 50 nm, and a height of 20 nm.

  FIG. 7B shows absorption spectra of the metal nanodots 1020 and 1021 in the air. When the peak wavelength of the metal nanodot 1020 and the peak wavelength of the metal nanodot 1021 are compared, the peak wavelength of the metal nanodot 1020 is lower. . Further, an absorption spectrum as shown in FIG. 7C is obtained from the spectrum patterns of the metal nanodots 1020 and the metal nanodots 1021. The target substance detection element immobilizes Anti-DNP capture molecules on the surface of the metal nanodots.

Next, the immunoassay in this example will be described. Anti-DNP is immobilized on the surface of the metal nanodots, and a specimen solution containing 1.5 × 10 ⁵ M DNBA is incubated on the immobilized target substance detection element to perform a binding reaction for 2 hours. Thereafter, when the sample solution is replaced with a phosphate buffer, the complex of DNBA and Anti-DNP shown in FIG. 5 is immobilized on the surface of the metal nanostructure.

  Next, a method for measuring the target substance will be described. The target substance detection apparatus shown in FIG. 2 measures the spectrum of the Raman scattering signal of the complex of antiDNP-DNBA.

  First, when the excitation light 205 is irradiated from the light source 202 to the metal nanodots 1020 and 1021, a strong evanescent electric field is induced around the metal nanodots 1020 and 1021.

  Subsequently, when the laser light near the absorption wavelength (near infrared band) of the metal nanodots 1020 and 1021 is irradiated from the light source 206 to the metal nanodots 1020 and 1021, Raman scattering occurs in the metal nanodots 1020 and 1021. The spectroscope 218 measures the spectrum of Raman scattered light that has been Raman scattered by the metal nanodots 1020 and 1021. According to the configuration of the present invention, it is possible to suppress the self-fluorescence due to the impurity impurities contained in the sample solution to a level that causes no practical problem, and to obtain Raman-scattered light.

  Referring to FIG. 7C, the absorption spectrum of metal nanodots 1020 and 1021 measured in the atmosphere, the absorption spectrum of metal nanodots 1020 and 1021 has an absorption peak near 1000 nm in the atmosphere. It is known from various experiments and examinations that the metal nanodots 1020 and 1021 have a characteristic of approximately 350 nm / index. The refractive index of the atmosphere is 1.0, and the refractive index when introduced into the sample solution is 1.4. Therefore, when actually introduced into the sample solution, the absorption peak wavelength of the metal nanodots 1020 and 1021 is calculated as 1000 + 350 × (1.4−1.0) = 1140 nm.

FIG. 8 is a diagram showing a Raman scattering spectrum in this example. In FIG. 8, the horizontal axis is the spectrum shift (cm ⁻¹ ), and the vertical axis is the intensity. In Figure 8, 1549cm ^-1, stretching vibration of a benzene ring in the vicinity of 1525 cm ^-1, stretching vibration of NO2 in the vicinity of 1338cm ^-1, it is possible to verify the stretching vibration near 839cm ^-1 in good S / N. In this case, excitation light, semiconductor laser: excitation is performed at 980 nm, and therefore, when described in terms of wavelength, there are peaks in the vicinity of 1155 nm, 1152 nm, 1128 nm, and 1067 nm. By analyzing these peaks, the amount of bonds can be measured by analyzing the bonds between molecules and the integrated intensity of the peaks.

  According to the present example, by arranging a plurality of metal nanostructures having different absorption wavelengths on the substrate, the Raman band can be substantially covered by the plasmon peak unique to the nanostructure. Thereby, the Raman scattering signal can be measured over a wide band.

(Example 3)
First, the configuration of the target substance detection element will be described with reference to FIG. 9A.

  The metal nanodots are four rectangular parallelepiped elements having anisotropy for defining an angle with incident light, and are arranged on a support.

  The metal nanodot 1021 is an element having a long side of 220 nm, a short side of 50 nm, and a height of 20 nm, and the metal nanodot 1020 is an element having a long side of 200 nm, a short side of 50 nm, and a height of 20 nm. The metal nanodot 1022 is an element having a long side of 180 nm, a short side of 50 nm, and a height of 20 nm, and the metal nanodot 1023 is an element having a long side of 160 nm, a short side of 50 nm, and a height of 20 nm.

  The angle θ between the principal axis direction of the metal nanodot and the deflection direction of the incident electric field is related by the equation (1). Here, i represents the type of rod group of metal nanodots, X1 is the length of the rod with the long side being short, and X (i) is the length of the main axis of the rod. For example, when the angle θ of the metal nanodot 1022 is calculated, the length of the short rod (metal nanodot 1023) is 160 nm, and the length of the long side of the metal nanodot 1022 rod is 180 nm. By substituting each value into Equation (1), the angle θ is calculated as 27.3 °.

  In the case of the target substance detection element shown in FIG. 9A, the metal nanodot 1023 having the shortest length in the major axis direction is arranged in parallel with the deflection direction of the incident electric field. The metal nanodot 1022 having the next shortest length in the major axis direction is arranged by rotating by 27.3 ° from the deflection direction. Further, the metal nanodots 1020 are arranged by rotating 36.9 ° from the deflection direction, and the metal nanodots 1021 having the longest flow in the major axis direction are arranged by rotating 43.3 ° from the deflection direction. The target substance detection element immobilizes Anti-DNP capture molecules on the surface of the metal nanodots.

  In addition, the angle θ between the principal axis direction of the metal nanodots and the deflection direction of the excitation light can be expressed by the equation (2) in consideration of the length in the minor axis direction. In this case, X (i) is the length in the major axis direction of the rod, Y (i) is the length in the minor axis direction of the rod, X1 is the length in the major axis direction of the rod having the shortest plasmon peak wavelength, and Y1 Is the length in the minor axis direction of the rod with the shortest plasmon peak wavelength.

Next, the immunoassay will be described. Anti-DNP is immobilized on the surface of the metal nanodots, and a specimen solution containing 1.5 × 10 ⁵ M DNBA is incubated on the immobilized target substance detection element to perform a binding reaction for 2 hours. Thereafter, when the sample solution is replaced with a phosphate buffer, a complex of DNBA and Anti-DNP is immobilized on the surface of the metal nanostructure as shown in FIG.

  Next, measurement of the target substance will be described. The target substance detection apparatus shown in FIG. 2 measures the Raman scattering spectrum of the complex of antiDNP-DNBA.

  First, when the metal nanodots 1020, 1021, 1022, and 1023 are irradiated with the excitation light 205 from the light source 202, a strong evanescent electric field is induced around the metal nanodots 1020, 1021, 1022, and 1023.

  Subsequently, when the laser light near the absorption wavelength (near infrared band) of the metal nanodots 1020, 1021, 1022, and 1023 is irradiated from the light source 206 to the metal nanodots 1020, 1021, 1022, and 1023, the metal nanodots 1020, 1021, and so on. Raman scattering occurs at 1022 and 1023. The spectroscope 218 measures the spectrum of Raman scattered light that has been Raman scattered by the metal nanodots 1020, 1021, 1022, and 1023. Since the metal nanodots 1020, 1021, 1022, and 1023 have different absorption wavelengths, the Raman scattering signal is more surely suppressed in order to suppress the self-fluorescence due to the impurity impurities contained in the sample solution to a level at which there is no practical problem. Can be measured.

FIG. 9B is a diagram showing a Raman scattering spectrum in this example. In Figure 9B, 1549cm ^-1, stretching vibration of a benzene ring in the vicinity of 1525 cm ^-1, stretching vibration of NO2 in the vicinity of 1338cm ^-1, stretching vibration near 839cm ^-1 is the best S / N. In this case, excitation light, semiconductor laser: excitation is performed at 980 nm, and therefore, when described in terms of wavelength, there are peaks in the vicinity of 1155 nm, 1152 nm, 1128 nm, and 1067 nm. By analyzing these peaks, the amount of bonds can be measured by analyzing the bonds between molecules and the integrated intensity of the peaks.

  According to the present example, when the major axis length and minor axis length of the plurality of metal nanodots are changed, the wavelength of the plasmon peak of the metal nanodot also changes. Therefore, when there are multiple wavelengths of the Raman band of the target substance, by adjusting the length of the major axis and the length of the minor axis of the metal nanodot, each position of the wavelength of the Raman band of the target substance and the metal nanodot Can match one of the plasmon peaks. At that time, if the length of the major axis or minor axis of the metal nanodot is changed, the intensity of the plasmon peak will change. In that case, the angle between the polarization direction of the incident light and the major axis direction of the metal nanodots may be changed so that the plasmon intensity of each metal nanodot becomes constant. According to this, even when a plurality of metal nanodots having different lengths in the major axis direction and the minor axis direction are used, a plurality of Raman bands of the target substance can be enhanced with uniform S / N.

Example 4
First, the configuration of the target substance detection element will be described with reference to FIG. 10A.

  The metal nanodot uses two rectangular parallelepiped elements having anisotropy for defining an angle with incident light. The metal nanodot 1020 has a long side of 200 nm, a short side of 50 nm, and a height of 20 nm, and the metal nanodot 1022 has a long side of 180 nm, a short side of 50 nm, and a height of 20 nm.

  Referring to FIG. 10B, Anti-IgG1301 capture molecules are immobilized on the surface of metal nanodots 1020, and anti-IgE1302 supplemental molecules are immobilized on the surfaces of metal nanodots 1022.

  Next, the immunoassay in this example will be described. Referring to FIG. 10B, a coating material for preventing nonspecific adsorption, for example, BSA (Bovine Serum Albumin) is adsorbed on the target substance detection element. As a buffer solution, a phosphate buffer solution (PBS (Phosphate buffered saline)) having a pH adjusted to around 7 is used, and a specimen solution containing IgG antigen 1303 and IgE antigen 1304 is introduced. When a binding reaction is performed for 2 hours after introduction of the sample solution, an antigen-antibody reaction as shown in FIG. 11A occurs. Thereafter, the sample solution is replaced with a phosphate buffer, and a sandwich assay is performed by introducing anti-IgG antibody 1305 and anti-IgE antibody 1306 labeled with a Raman-active molecule (see FIG. 11B).

  Next, a method for measuring the target substance will be described. The target substance detection apparatus shown in FIG. 2 measures the Raman scattering spectrum of the antibody-antigen complex labeled with DNBA.

  First, when the metal nanodots 1020 and 1022 are irradiated with the excitation light 205 from the light source 202, a strong evanescent electric field is induced around the metal nanodots.

  Subsequently, when the laser light near the absorption wavelength (near infrared band) of the metal nanodots 1020 and 1022 is irradiated from the light source 206 to the metal nanodots 1020 and 1022, Raman scattering occurs in the metal nanodots 1020 and 1022. The spectroscope 218 measures the spectrum of Raman scattered light that has been Raman scattered by the metal nanodots 1020 and 1022. According to the configuration of the present invention, it is possible to suppress the self-fluorescence due to the impurity impurities contained in the sample solution to a level that causes no practical problem, and to obtain Raman-scattered light.

FIG. 12 is a diagram showing the spectrum of the Raman scattering signal in this example. In Figure 12, 1549Cm ^-1 corresponding to the peak of the plasmon absorption spectrum, the stretching vibration and 839cm stretching vibration in the vicinity of ^-1 benzene ring in the vicinity of 1525 cm ^-1 becomes the best S / N. According to this example, the amount of antigen bound to the antibody can be quantitatively measured by measuring the plasmon peak.

(Example 5)
Next, an example in which an immune response is measured without using a Raman active molecule will be described in detail.

  The configuration of the target substance detection element will be described with reference to FIG. 7A. The metal nanodot uses two rectangular parallelepiped elements having anisotropy for defining an angle with incident light. The metal nanodot 1020 is an element having a long side 200, a short side 50 nm, and a height 20 nm, and the metal nanodot 1021 is an element having a long side 220, a short side 50 nm, and a height 20 nm.

FIG. 7B shows absorption spectra of the metal nanodots 1020 and 1021 in the air. When the peak wavelength of the metal nanodots 1020 and the peak wavelength of the metal nanodots 1021 are compared, the peak wavelength of the metal nanodots 1020 is lower. . Further, an absorption spectrum as shown in FIG. 7C is obtained from the spectrum patterns of the metal nanodots 1020 and the metal nanodots 1021. Anti-IgG1301 having a concentration of 2.0 × ^{10 −10} mol / L is immobilized on the surfaces of the metal nanodots 1020 and 1021.

  Next, the immunoassay in this example will be described. BSA (Bovine Serum Albumin) is adsorbed as a coating material for preventing non-specific adsorption on a target substance detection element having a capturing body immobilized on the surface. As a buffer solution, a phosphate buffer solution (PBS) whose pH is adjusted to around 7 is used, a solution containing IgG antigen 1303 is introduced, and a binding reaction is performed for 2 hours.

  Next, the configuration of the detection apparatus in the present embodiment will be described with reference to FIG. The detection apparatus includes a support 101, metal nanodots 1020 and 1021, light sources 601 and 610, a filter 602, a collimator lens 603, a prism 604, an objective lens 607, a dichroic mirror 608, a collimator lens 609, a total reflection mirror 611, a lens 612, An aperture 613, a lens 614, an aperture 615, a notch filter 616, and a spectroscope 617 are provided. With such a detector, the Raman scattering spectrum of the complex of anti-IgG and IgG is measured.

  First, when the metal nanodots 1020 and 1021 are irradiated with the excitation light 205 from the light source 601, a strong evanescent electric field is induced around the metal nanodots 1020 and 1021.

  Subsequently, when the laser light near the absorption wavelength (near infrared band) of the metal nanodots 1020 and 1021 is irradiated from the light source 610 to the metal nanodots 1020 and 1021, Raman scattering occurs in the metal nanodots 1020 and 1021. The spectroscope 617 measures the spectrum of Raman scattered light that has been subjected to Raman scattering by the metal nanodots 1020 and 1021. According to the configuration of the present invention, it is possible to suppress the self-fluorescence due to the impurity impurities contained in the sample solution to a level that causes no practical problem, and to obtain Raman-scattered light.

  FIG. 7C is a diagram obtained by measuring an absorption spectrum in the atmosphere of the metal nanostructure. In FIG. 7C, the horizontal axis represents wavelength (nm) and the vertical axis represents absorbance (OD). The absorption spectrum of the metal nanodots 1020 and 1021 has an absorption peak near 1000 nm. It is known from various experiments and examinations that the metal nanodots 1020 and 1021 that are metal nanostructures have a characteristic of approximately 350 nm / index. The refractive index of the atmosphere is 1.0, and the refractive index when introduced into the sample solution is 1.4. Therefore, when the sample solution is actually introduced, the wavelength of the absorption peak of the metal nanodots 1020 and 1021 is calculated as 1000 + 350 × (1.4−1.0) = 1140 nm.

  FIG. 14 is a diagram showing a Raman scattering spectrum in this example. The Raman scattering spectrum 700 is a Raman scattering spectrum when only anti-IgG exists on the surface of the metal nanodot. A Raman scattering spectrum 701 is a Raman scattering spectrum obtained from a complex of anti-IgG and IgG. The spectrum changes greatly before and after the reaction, and the concentration of the antigen can be measured by using these spectra.

  According to the present invention, the concentration of a marker related to a disease in serum, the blood concentration of a drug administered, or the blood concentration of a metabolite is examined to help diagnose a disease or determine a medical treatment policy. be able to.

It is a figure which shows the board | substrate for element formation, and a target substance detection element. It is a figure explaining the structure of a target substance detection apparatus. It is a figure explaining the process which produces a target substance detection element. It is a figure which shows the metal nanodot by a SEM (Scanning Electron Microscope) image. It is the figure which measured the absorption spectrum in air | atmosphere in a metal nanostructure. It is a figure when the composite_body | complex of DNBA and Anti-DNBA is fix | immobilized on the surface of a metal nanostructure. 2 is a diagram showing a Raman scattering spectrum in Example 1. FIG. It is a figure explaining the structure of the element for target substance detection. It is a figure which shows the absorption spectrum of the metal nanodots 1020 and 1021 in the air. It is the figure which piled up the absorption spectrum of metal nanodot 1020,1021 in the air. 6 is a diagram showing a Raman scattering spectrum in Example 2. FIG. It is a figure explaining the structure of the element for target substance detection. 6 is a diagram showing a Raman scattering spectrum in Example 3. FIG. It is a figure explaining the structure of the element for target substance detection. It is a figure which shows a state when the capture body is fix | immobilized on the surface of metal nanodot. It is a figure which shows a state when an antigen antibody reaction arises. It is a figure which shows a state when a sandwich assay is performed. It is a figure which shows the spectrum of the Raman scattering signal in the present Example 4. It is a figure explaining the structure of the detection apparatus in the present Example 5. FIG. It is a figure which shows the Raman scattering spectrum in the present Example 5.

Explanation of symbols

DESCRIPTION OF SYMBOLS 101 Support body 102,1020,1021,1022,1023 Metal nanodot 103 Capture body 202 Light source 203 Filter 204 Condensing lens 205 Excitation light 206 Light source 207 Collimator lens 208 Dichroic mirror 209 Objective lens 210 Total reflection mirror 211 Lens 212 Aperture 213 Lens 214 Aperture 215 Notch filter 218 Spectrometer 401 Support 402 Au layer 403 Resist layer 601 Light source 602 Filter 603 Collimate lens 604 Prism 607 Objective lens 608 Dichroic mirror 609 Collimate lens 610 Light source 611 Total reflection mirror 612 Lens 613 Aperture 6 614 Lens 613 Lens Filter 617 Spectrometer 700,7 1 Raman scattering spectra 1301 Anti-IgG
1302 Anti-IgE
1303 IgG antigen 1304 IgE antigen 1305 Anti-IgG antibody labeled with Raman-active molecule 1306 Anti-IgE antibody labeled with Raman-active molecule

Claims (6)

A target substance detection element for detecting a target substance in a specimen,
A support;
Metal nanodots present on the surface of the support;
A capture molecule that is present on the surface of the metal nanodot and captures a target substance in the specimen;
The metal nanodot has n plasmon resonance spectral peaks,
The peak wavelength of the n-th largest peak among the peaks of the Raman band of the target substance in the sample and the peak wavelengths of the n peaks of the metal nanodots correspond to each other in the vicinity. A target substance detection element.   2. The target substance detection element according to claim 1, wherein the metal nanodot is composed of a plurality of types of metal nanodots having different absorption wavelengths.   The target substance detection element according to claim 1, wherein the metal nanodot has anisotropy. The metal nanodots are composed of a plurality of types of rod groups having different shapes, and an angle θ between a main axis direction of the rod and a deflection direction of excitation light of the rod has the following relationship: The target substance detection element according to claim 3.

(I is the type of rod group, X (i) is the length in the major axis direction of each type of rod, Y (i) is the length in the minor axis direction of each type of rod, and X1 is the wavelength of the plasmon peak. (The length in the major axis direction of the short rod, Y1 represents the length in the minor axis direction of the rod with the shortest plasmon peak wavelength) The target substance detection element according to claim 1,
A first light source that emits light for inducing an evanescent electric field in the vicinity of the metal nanodots of the target substance detection element;
A second light source for irradiating the metal nanodots with light for causing Raman scattering;
And a spectroscope for detecting the Raman scattered light enhanced by the metal nanodots.   6. The target substance detection apparatus according to claim 5, wherein the wavelength of the light from the second light source is a wavelength that does not excite the autofluorescence of impurities contained in the specimen.

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