JP3978498B2 - Method of forming metal nanotriangular prism array substrate for single molecule Raman spectroscopy and single molecule analysis method thereby - Google Patents

Description

  The present invention relates to a method for analyzing a single molecule and its substrate using enhancement of a Raman scattering spectrum by a strong electric field on a metal surface having a nanostructure, in particular, a triangular prism formed by nanobead lithography and a similar shape in single molecule measurement. Related to the creation method.

  The present invention relates to a method for creating a structure having a high optical activity on a surface. By utilizing the activity of the surface, a substance related to biochemistry can be detected with a single molecule sensitivity, and quantitative and qualitative analysis can be performed. Includes devices. More specifically, the present invention relates to a method of regularly arranging nanometer-scale fine particles and Raman scattering using strong electric field formation by surface plasmons on the surface of a metal nanostructure obtained by using the monolayer film as a template. The present invention relates to the measurement principle of a single molecule sensitivity of a spectrum and its device (substrate). The device also includes methods for measuring single molecule Raman scattering signals of biochemically related substances and thereby analyzing the state of the target molecule, and methods for measuring single molecules in a wide range of other fields.

  Conventionally, as a method for forming a single particle layer film, a method for forming a single particle layer film, a multi-particle layer film by an advection accumulation method, a method for forming an LB film, and the like are known. Among these, the advection accumulation method is based on the principle that the glass is permeated for a long time in a solution in which spherical nanoparticles that can be dispersed for a long time in a solvent (mainly water) are suspended and pulled up. By using an improved glass surface treatment (specifically, using casein as glue), controlling temperature, humidity, particle concentration, glass pulling speed, etc. A single particle layer film and a multi-particle film are formed on a glass substrate. When a single particle layer film is to be formed from a solution suspended by drying as seen in Non-Patent Documents 1 and 2, in addition to the single particle layer film part, a part lacking many particles Is seen. In addition, there is a method in which the surface of a glass is treated with casein, as reported in Non-Patent Document 3, and used as a paste between nanoparticles suspended in a solution and glass. As shown in this report, many defects are observed, and the process of creating a single particle layer film is complicated, such as the work of attaching the casein to the glass substrate. Further, as seen in Non-Patent Document 4, as a method for producing a single particle layer film using the self-accumulation force of molecules and the force of drying of a solvent, a glass substrate is moved at a constant speed and is moved to the surface. Some are made by pouring nanoparticles from a suspension and controlling the solvent flow and drying rate. However, this method requires a device for controlling the distance from the surface of the glass and the movement of the glass serving as the substrate, and the portion to be a single particle layer film is not a single particle layer but a two particle layer. There are also three-particle layers.

  The LB film creation method is not simply a method of pulling up the glass substrate from the suspension, but mainly by developing a hydrophilic or hydrophobic substance in which an organic solvent is dissolved on the surface of the aqueous solution and controlling the surface entropy. This is a well-known method for producing a thin film for transferring a single particle layer film on a water surface onto a solid substrate and repeating it to accumulate it in a multi-particle layer. Although this method is a very excellent method for forming a thin film, the state of the thin film strongly affects the wettability of the surface of the substrate. However, it is still difficult to control this wettability due to the intertwining of complex factors such as surface roughness and chemical interaction with the surface. In addition, when using a suspension sphere of nanoparticles suspended in an aqueous solution, its own weight and chemical surface condition factors are added, which increases the reproducibility and continuity of the monolayer film. Problems come out. Even if it can be controlled, the repulsive force or attractive force between the nanoparticles will affect the formation of the single particle layer film on the glass substrate, and as a result, a multilayer film will be formed with the defective part or part of the single particle layer film. End up.

By the way, a single particle layer film was created by some method using a turbid liquid of nanoparticles, and an array of metal having a nanostructure on the surface was created by a deposition method, and the characteristics of the metal nanostructure were examined. Examples can be found in Non-Patent Documents 6 and 11. In these, the stability of the surface of the metal nanostructure at the time of surface enhanced Raman scattering measurement is investigated by cyclic voltammentry, and the metal nanostructure interval is tilted in the direction to the deposition source of the substrate at the time of deposition. Attempts have been made to control the structure spacing, increase the coupling of surface plasmons localized there, and obtain a large Raman enhancement. However, it is in principle difficult to obtain a single-molecule sensitivity Raman signal intensity by such an attempt. This is because, according to Non-Patent Document 7, it is not possible to obtain a huge enhancement of 10 ¹⁰ or more due to localized surface plasmons unless the above-described method has extremely sharp joints between metal nanostructures. This is because it is clarified by electromagnetic calculation as shown. On the other hand, in the metal nanostructure formed by controlling the deposition conditions using a single-layer film of nanoparticles as a template, the electric field at the edge of the triangular prism structure is particularly strong, and the effect of surface plasmon is extremely efficient. Therefore, it is expected that the Raman scattering signal intensity is enhanced to 10 ¹⁰ or more. Thereby, it is considered possible to measure the Raman spectrum of the adsorbed species with single molecule sensitivity.

  The potential for single molecule measurements has been tested in many ways. Among them, attempts to measure a single molecule using a spectroscopic method include a method using a fluorescence method and a method using Raman scattering, particularly surface-enhanced Raman scattering. Measurement of a single molecule by a fluorescence method is used in many cases such as measurement of a single molecule, although it shows strong fluorescence such as rhodamine 6G, and observation of a labeled DNA under a microscope. However, in the fluorescence spectrum, the characteristic peak is broad, and in many cases, it is not suitable as a method for analysis at the single molecule level of unknown substances, unless it is previously labeled as a marker.

On the other hand, when using Raman scattering, when incident light hits a molecule, the reflected light reflects the intrinsic energy state based on the bond within the molecule or between molecules (scattered light energy = incident light energy ± vibration energy). Are generated depending on the number of intramolecular bonds. That is, since a plurality of characteristic spectral peaks indicating the structure of the substance and its interaction with the surroundings are obtained, it is superior to the fluorescence method for qualitative and quantitative analysis of chemical species. However, Raman scattered light is inherently weaker than fluorescence, so it is not suitable for measurement with a single molecule. However, it has been reported in Non-Patent Document 8 by several groups, for example, Nie et al. That measurement by a single molecule is possible by utilizing surface-enhanced Raman scattering. Surface enhanced Raman scattering is an enhanced Raman scattering effect that occurs when a molecule discovered by Richard van Dyne in 1974 is attached to a rough metal surface. Its enhanced intensity of the signal may up to as high as 10 ⁶ as an average of the number of molecules to be measured simultaneously. Recent developments in probe microscopes and high-sensitivity photodetectors have made it possible to detect signals only from metal particles having an optimal shape that gives a large signal intensity. Thereby, a huge Raman signal enhancement of 10 ¹⁰ to 10 ¹⁴ has been reported.

In the measurement of single molecules using this surface-enhanced Raman scattering, a phenomenon called “Blinking” (a phenomenon in which the Raman signal intensity is large and repeatedly changes over time, the heat on the metal surface of one adsorbed molecule) Observed to be due to diffusion). This report on blinking is a single molecule measurement by surface-enhanced Raman scattering first reported in Non-Patent Document 8. Nie et al observed a surface enhanced Raman scattering of rhodamine 6G in silver particles table surface produced from chemical reduction, that is a signal from a single molecule of a silver surface of a sphere-shaped grains terminal of a diameter of about 150 nm, It was confirmed from the dependency of the signal intensity on the amount of adsorption and the deflection characteristics.

As for blinking, the optimum size as in Non-Patent Document 9 depends on the excitation wavelength. Further, as in Non-Patent Document 10, in addition to surface plasmon, there is an electron transfer between the metal and the adsorbed species. It is pointed out that it contributes. Although the mechanism of complete enhancement has not been completely clarified, various studies have been conducted on its origin, and various findings are being obtained.
However, the surface-enhanced Raman scattering metal nanoparticles created in these reports are simple ones in which silver colloidal particles obtained by reducing silver nitrate or the like with citric acid are fixed on a substrate, and have various shapes and sizes. -Consists of agglomerated state. From the enormous number of particles, selecting a very small number of particles having an optimal shape and using them for measurement is inefficient in terms of application and is not practical. Furthermore, in such a method, (1) a metal nanostructure having an optimum shape for surface-enhanced Raman scattering cannot be efficiently created. In addition, (2) even if it can be made, it cannot be arranged in two dimensions, and there are some disadvantages to use it as a single molecule analysis device.
Langmuir, Vol. 8, 1992, pp 3182-3190 Nature, 361, 1993, p26 Langmuir, Vol. 10, 1994, pp 432-440 Langmuir, Vol. 14, 1998, pp 6441-6447 Journal of Physical Chemistry B, Vol. 103, 1999, pp 9846-9853 Journal of Physical Chemistry B, Vol. 106, 2002, pp 853-860 Journal of Physical Chemistry B, 107, 2003, pp 7607-7617 Science, 107, 1997, pp 1102-1106 Journal of the American Chemical Society, Vol. 121, 1999, pp 9208-9214 Journal of the American Chemical Society, Vol. 121, 1999, p9932-9939 Journal of Physical Chemistry B, 107, 2003, pp 7426-7433

Although it is possible to produce a nanoparticle monolayer film using a solution in which nanoparticles are suspended by a conventional method, it is difficult to produce it in a wide area, and the associated equipment and devices are complicated and troublesome. It is a thing. Furthermore, although the surface enhanced Raman scattering has been confirmed in the structure of the prepared metal triangular prism, the edge of the nano triangular prism formed probably due to insufficient control of the deposition conditions has a radius of curvature of about 10 nm. For this reason, only an intensification insufficient for detecting single molecules of about 10 ⁵ to 10 ⁷ is obtained. In addition, in order to analyze a polysaccharide sample in as short a time as possible in connection with high-sensitivity analysis such as biochemistry, a two-dimensional array as wide as possible is required. Therefore, the purpose here is (1) to form a single-layer film of nanoparticles in an orderly array (1000 μm × 100 μm) as wide as possible, and (2) to use it to sharp edges by controlling the deposition conditions Forming a metal nano-triangular prism structure array containing (3), thereby obtaining a Raman enhancement intensity of 10 ¹⁰ or more, thereby efficiently performing surface-enhanced Raman scattering of a single molecule of a wide range of chemical species present on the nanostructure surface It is to provide a method for detection and qualitative analysis.

The present invention
(1) In a method for producing a substrate having a metal nano-triangular prism structure array formed by forming a close-packed monolayer film of nanoparticles on a substrate, vacuum-depositing a metal from the substrate, and washing away the nanoparticles. The formation of a single layer film in which the nanoparticles are arranged two-dimensionally uses two glass substrates having different affinities for the nanoparticles, and one of the substrates having a high affinity is allowed to stand horizontally and its surface A nanoparticle suspension is dropped onto the substrate, and the other relatively low affinity substrate is placed on the spacer against one end of the substrate and placed obliquely, and the humidity is controlled to 60% or more. A method for producing a substrate having a metal nano-triangular prism structure array, characterized by being performed by drying with
(2) The method for producing a substrate according to (1), wherein the two substrates are both glass, and the hydrophilic strength of each glass surface is different, and
(3) A single molecule state characterized by measuring a surface-enhanced Raman scattering spectrum and its blinking in a metal nanotriangular prism array using the substrate prepared as described in (1) or (2) An analysis method and a substrate thereof are provided.

Terms used in the present invention are defined.
As used herein, “nanoparticle” refers to a sphere having a radius (r) of 1 nanometer <r <1000 nanometer (1 nanometer = 10 ⁻⁹ meter).
As used herein, “blinking” indicates that the intensity of a surface-enhanced Raman scattering signal derived from a single molecule repeatedly flashes with a period of several hertz over time.
As used in this specification, an “array” is a structure in which nano-scale three-dimensional metal structures (including triangular prisms) created by lithography using nanoparticles are regularly arranged, or part of the structure is interrupted. 2 shows a structure in which two or more nanoscale three-dimensional structures are aligned.
As used herein, a “flat portion” is a portion where metal is deposited on a portion other than the array where no template substrate of nanoparticles is present.

  According to the present invention, it is possible to easily and easily create a device capable of generating a strong electric field, and to perform state analysis at a single molecule level such as a biomolecule.

One preferred embodiment of the present invention is that (1) a single-particle layer two-dimensional close-packed structure of nanoparticles is formed on a suitable substrate surface, (2) a metal is deposited from above, and a giant enhancement strength is formed in the gap. And (3) forming a single-molecule Raman spectroscopic substrate having an array of metal nanostructures by washing away unnecessary nanoparticles after that.
(4) In actual analysis, various methods, such as the simplest method, on the surface of the substrate, a sample solution whose concentration is adjusted in advance so as to obtain a single molecule level coverage per nanostructure is applied to the substrate surface. After dropping and drying, the sample is placed under a microscope to measure the Raman scattering of the sample.
At this time, for the purpose of (1), by controlling the dynamic affinity between the nanoparticles and the substrate by controlling the surface treatment of the substrate and the solvent evaporation mechanism / deposition rate, the largest possible close-packed structure is achieved. It is preferable to form a single particle layer. Regarding (2), in order to form a sufficiently sharp edge in the metal nanostructure, it is preferable to control conditions such as the spatial arrangement of the substrate and the vapor deposition source, the vapor deposition technique, the speed and the degree of vacuum. Furthermore, (3) in order to peel only the nanoparticles from the substrate, it is preferable to optimize the solvent type, ultrasonic cleaning output, temperature, time, and the like.

Moreover, the specific example of the preparation method of the single particle layer film | membrane of a nanoparticle is as follows. (1) Prepare two flat plates having different affinity for nanoparticles by surface treatment or the like. (2) One substrate having high affinity is left still in advance, and an appropriate amount of nanoparticle suspension is dropped. A substrate having different affinity for nanoparticles is placed on the substrate, and the substrate is placed diagonally with a spacer on the opposite side.
Various materials can be used as the substrate that can be used at this time, but a substrate that has as little influence as possible, such as not emitting scattered light or fluorescence that interferes with the subsequent Raman scattering measurement, is desirable. One of them is many kinds of glass. Further, the second covering plate needs to be sufficiently stiff so as not to lose the surface tension of the nanoparticles in order to stabilize the single particle layer structure. Nanoparticles sandwiched between two plates are dried as they are in a device such as a desiccator with controlled humidity. In this process, it is necessary to effectively use the surface tension and capillary action accompanying the drying of the solvent so as to make the single particle layer structure as large as possible. Therefore, the humidity is preferably about 60% or more for the purpose of suppressing the drying rate.
The nanoparticle monolayer film produced by this method can be spread over a wide area of 1000 μm × 1000 μm. Furthermore, as described above, the creation method is simple. The formed nanoparticle two-dimensional array structure can be obtained by feeding back to the preparation method by measuring the structure at the nanoscale with an atomic force microscope in addition to the optical microscope. It helps to form more widely.

In the array of metal nanostructures produced according to the present invention, the triangular prism-like nanostructure predicted by theoretical calculation has a sharp edge, so that the upper surface (cross section) of the triangular prism is preferably as smooth as possible. Since the metal thin film generally formed by vapor deposition consists of nanoparticle aggregates, the deposition method and the arrangement of the vapor deposition source and the substrate in the apparatus, the vapor deposition conditions are controlled, and a metal film that is as smooth as possible is formed. Moreover, it is preferable to have sufficient adhesion between the formed metal structure and the substrate.
As a result of preliminary investigations on these, it is necessary to use a resistance heating method, and use a material that is sufficiently far from the deposition source so that the flow (flux) of metal atoms and clusters during deposition is as parallel as possible. Good. Further, according to the vapor deposition apparatus having such characteristics, an enhancement effect can be expected with respect to the intensity of the electric field in the array, and the planar portion of the triangular prism constituting the array made thereby is relatively flat (height difference ± 3 nm). It is suitable for creating an array. Also includes metal capable of forming a single molecule SERS for nano-array structure by vacuum deposition is not particularly limited, gold, silver, other platinum copper, a transition metal such as nickel. As a vapor deposition method, a conventional method can be used, but resistance heating, sputtering, electron beam vapor deposition and the like are preferable.

A spectroscopic image device for measuring blinking of surface-enhanced Raman scattering is based on a microscope using an objective lens or the like and is provided with a two-dimensional detector such as a CCD (charge coupled device) for image observation. In front of the detector, Rayleigh light (Rayleigh light, elastic scattered light having the same wavelength as the incident light, which has an intensity of 10 ¹⁰ or more of Raman scattered light, and particularly weak interference with signal measurement. Use an optical filter that efficiently removes. In addition, in order to obtain detailed information such as identification of chemical species, it is necessary to perform spectrum measurement of surface enhanced Raman scattering. For this purpose, a measurement system comprising a spectroscope and a two-dimensional photodetector optically connected to the microscope is constructed. As a result, it is possible to measure the surface-enhanced Raman scattering spectrum together with the detection of blinking, and to analyze the state of the target chemical species at the single molecule level.

  Hereinafter, the contents of the present invention will be specifically described based on examples. In addition, this invention is not limited about a following example.

Suspensions of polystyrene nanoparticles with a diameter of 500 nm (DUKE Scientific, USA) were diluted in stock solution concentration (1 g / ml) by half with distilled water. A commercially available microscope slide glass is thoroughly washed with a detergent and then further thoroughly washed with distilled water in an ultrasonic cleaner. Next, as shown in FIG. 1, 30 μl of the suspension 2 is dropped onto the slide glass that becomes the glass substrate 3 (FIG. 1A), and a glass having hydrophilicity different from that of the glass substrate 3 is formed thereon. Cover with the substrate 4 (the glass substrate 3 is immersed in a dilute alkaline aqueous solution for about 10 minutes, taken out, and then washed once or twice with pure water). In that case every spacer 1 (that cut 1mm cover glass), covered with a glass substrate 4 so as to prop up against one end of the glass substrate 4 in space Sa 1 (FIG. 1 (B)). By elevating one side of the glass substrate 4 with the spacer 1, the evaporation rate of water as a solvent can be controlled (FIG. 1C). It is allowed to stand for about 12 hours in a desiccator whose humidity is controlled to 65 to 80 % and dried. Then, as shown in FIG. 1D, the single particle layer film 5 can be formed in a wide area. By this method, it was clearly confirmed by visual observation and an optical microscope that a single particle layer film of nanoparticles was formed on the glass substrate. When both this is performed in those with the power of the same parent miscible, it is not successfully created a single particle layer film. Moreover, as the size of the single particle layer film made of the substrates having different affinities, some of the size reached about 1 mm × 500 μm. Furthermore, when a 2M pixel digital CCD camera of Sony DSC-50C is installed in an optical microscope (Olympus) having an objective lens with a numerical aperture (NA) 0.9 of 100 times magnification, an image thereof is obtained. The color of the part with the structure of the single particle layer film gave a distinctly different color compared to the place where it was not.

  In order to show this clearly, the result of measuring the scattering spectrum of the single particle layer film surface is shown in FIG. This scattering spectrum is obtained by drawing the scattering spectrum of the glass portion as a background. A characteristic spectrum with a peak around 450 nm was obtained. This is the same phenomenon as that recently attracted attention as a diffraction phenomenon caused by the two-dimensional arrangement structure of nanoparticles (photorefractive effect), but in this case as well, the approximate position of the single particle layer film with the naked eye is known. Available for.

  Moreover, as a result of observing the single particle layer film of the nanoparticles using an atomic force microscope (AFM), it was confirmed that a very close and wide array structure was formed. Further, it was found that the gap (defect) portion of the bead structure, which was not apparent with the optical microscope, could be clearly resolved in the AFM image. Furthermore, there were very few defects. Here, the atomic microscope (AFM, Digital Instrument CA USA) was used in the tapping mode, and the scan width was in the range of 50 μm × 50 μm. The resolution is about 3 nm in the height direction (Z-axis), and the resolution in the horizontal direction is about 5 nm. The software attached to the AFM apparatus was used for processing the measured image. The spread angle between the slide glasses used in the formation of the nanobead single particle layer was about 3.18 ° when a 1 mm thick spacer was used.

A silver nanosubstrate was prepared by the following method using the single particle layer film using the 500 nm nanoparticles (nanobeads) prepared above.
Silver was deposited on the single particle layer film using a vacuum deposition apparatus (Varian turbo molecular pump, multipurpose stainless steel chamber having a rotary pump, with a resistance heating power source, or with a sputtering power source). The film thickness was measured at any time with a crystal oscillator type film thickness monitor (INFICON, East Syracuse, NY), and the total film thickness was 500A. The polystyrene nanobeads used as a template for the silver deposition film were removed by ultrasonic cleaning after the silver deposition.
Ethanol was used as a washing solution, and washing was performed at 100 kHz (50 W) for 3 minutes. During cleaning, it was confirmed with the naked eye that the beads were peeled off from the deposition surface.

  The array after washing the nanobeads is shown in FIG. A part of it was washed away by ultrasonic cleaning, but the structure of the array could be confirmed. It is also a wide area and extends at least 30 μm × 30 μm. In addition, it was found that the triangular prism structure was present at the apex of the hexagon by the nanoparticle template, and this triangular prism had a height of 80 nm and a length from the hem to the hem of 450 nm (FIG. 4). . FIG. 5 shows a three-dimensional view of the silver array.

Next, rhodamine 6G was dropped on the surface of the array, and blinking of the surface enhanced Raman scattering was observed. For the measurement of blinking, a micro Raman spectrometer with a CCD attached to a Renishaw spectrometer was used. The objective lens is Olympus N.P. A = 0.55 was used, and the intensity of the laser was about 30 μW on the surface of the sample. The image of the surface enhanced Raman scattering blinking was measured by using a 30-frame / second CCD camera combined with an image intensifier. The amplification factor of the image intensifier at that time was 560. Images taken with the CCD are A / D converted and stored in a computer. Surface enhanced Raman scattering from the blinking of rhodamine 6G is shown in FIG. This spectrum does not occur constantly as described above, but is the value of integration for 10 seconds. However, blinking may not occur even during the 10 seconds, and in this case, surface enhanced Raman scattering could not be measured. Rhodamine 6G having a concentration of 10 ⁻¹⁰ M was dropped on each part by 0.5 μl and allowed to stand for a while. FIG. 6B shows the background.

In order to compare the frequency of the blinking effect, a comparison was made between the portion of the array and the portion where silver was deposited as a particle aggregate (hereinafter referred to as a continuous film) having a diameter of 50 to 100 nm on a bare glass substrate. . The continuous film is obtained by depositing silver on the surface of glass by 50 nm. The results are shown in FIG. The area of about 10 μm × 10 μm was trimmed so as not to overlap each other for the array part and the silver continuous film part, and the number of blinking in the area was statistically analyzed. Considering optical resolution (about 600 nm = 0.61λ / NA, λ = wavelength), the inside is divided into squares of 4 pixels × 4 pixels. Using this as an observation unit, the presence or absence of blinking in this section was measured. At this time, from the characteristics of the microscopic optical system and the size per CCD pixel, the size on the sample corresponds to 0.347 μm / pixel, so 4 pixels corresponds to twice the theoretical resolution (about 600 nm). When this is standardized by 7 × 7 sections (49 observation areas are created in this area), the overall size is 9.716 μm × 9.716 μm. Evaluate the presence or absence of blinking in this area for 10 seconds (the area that showed Blinking even once in 10 seconds (4 × 4 pixels) is counted as 1. The number of such areas in 10 seconds Count). When the whole is blinking, the “number of locations where blinking has occurred” as shown on the horizontal axis of FIG. The concentration of the sample dropped on the surface is 10 ⁻¹⁰ M, and the number of sample molecules in the range of 10 μm × 10 μm is 38.25 when the spread of the surface is assumed to be a circle of equal concentration of 1 cm. It is estimated.

For a plurality of array structure samples formed in exactly the same manner, the total number of portions trimmed in the range of about 10 μm × 10 μm was 104 (a total of 104 samples of 10 μm × 10 μm were sampled including different samples). ). When comparing the array structure part and the continuous film part, the part where the blinking cannot be observed at all in the range of about 10 μm x 10 μm (corresponding to “number of points” on the horizontal axis in FIG. 7) is the surface of the continuous film. About 37% (= 38/108) and about 9.6% (= 10/108) in the array part. Furthermore, when the number of locations where blinking occurred at 5 locations or more (value 5 on the horizontal axis in FIG. 7) was compared, 25.9% (28/108) in the continuous film and 60.5% (66 in the array portion) / 108), and the frequency of the array portion is about 2.3 times that of the flat portion. This means that it is approximately 2.3 times more advantageous for measuring surface enhanced Raman scattering. In the total number of blinking (where blinking occurs X _n (n = 1, 2,...), Frequency Y _n is ΣX _n × Y _n ), 325 in the flat portion and 653 in the array portion It was a place.

Assuming that a complete array is formed over the entire observation area, the ratio of the array to the entire substrate (= total cross-sectional area occupied by one metal nanostructure / total cross-sectional area of the substrate) is 9.35%. It becomes. That is, in the portion where blinking is observed, the metal nanostructure is about 21 times more efficient per unit area than the continuous film.
Further, the ratio of the array structure formed for the sample used in the experiment from the AFM image of FIG. 3 (= area fraction of the actual silver triangular prism structure / total area of the substrate) is 12%, which is a theoretical value. It is rather big compared to. This is because, as shown in FIG. 3, the triangular prism structure is partially washed away, and the structure to be elongated and continuous with the region to be originally washed away remains. Based on theoretical calculations, the area of sharp edges in the measured shape is much smaller than the theoretical value (according to the number of edges, the measured value / theoretical value is ½ or less). Nanotriangular structures that work effectively to provide sensitivity are considered to be ½ or less. Therefore, it is considered that the blinking expression efficiency is greatly improved (twice or more) by controlling the washing conditions of the nanoparticles. Further, since the “continuous film portion” used this time is not subjected to any heat treatment such as annealing, a particulate silver aggregate having a diameter of 50 to 100 nm is formed. It is widely known from theoretical calculations and experiments that, for example, a perfectly flat sample cannot obtain a huge enhancement at a portion other than its edge (for example, Journal of Physics: Condensed Matter 1992, Vol. 4, page 1143). ).

Therefore, in order to more accurately evaluate the efficiency with which single-molecule sensitivity can be obtained in the array portion formed this time, the 50 nm-thick silver continuous film formed on the glass substrate is flattened by annealing, and this film is The dye samples were prepared under the same conditions as described above, and surface-enhanced Raman scattering measurement was thereby performed. The result is very clear, the silver continuous film obtained by 2 hours annealing at 200 ° C., the blinking conditions used for measurement on this array, blinking from R6G film is not observed at all, the surface enhanced Raman scattering signal was not obtained. It was confirmed by AFM measurement that the particle size was increased about 2-3 times by annealing of the continuous film, and the sharp edge between the particles before annealing was changed to a smoothly connected continuous film. From this, it is considered that the blinking that had occurred at the junction was completely suppressed because the area of the nanoparticle junction before annealing was significantly reduced by the heat treatment. As described above, the metal nano-triangular prism structure works extremely effectively for single-molecule sensitive surface-enhanced Raman scattering detection, and the two-dimensional array structure is formed here and can be used as a single-molecule surface-enhanced Raman scattering substrate. I found.

  As described above, it is possible to create a metal array using a nanoparticle monolayer film prepared by a simpler method, and to efficiently measure the effect of surface enhanced Raman scattering on the surface. did it. Further, this blinking effect is an effect from a single molecule, and as a result, a relatively small dye molecule, not a polymer, can be measured at the single molecule level.

  The present invention is not limited to the field of biochemical analysis, but includes analysis of constituent molecules for the realization of single molecule devices and nanochemical factories formed by bottom-up nanotechnology, catalyst design from the single molecule level, This is a useful basic technology in a wide range of fields where functions and structures need to be controlled at the molecular level.

It is explanatory drawing of the method of arranging a nanoparticle on a single particle layer film, and its simple apparatus. It is a spectrum of the scattered light of the single particle layer film | membrane of the nanoparticle produced in the Example. It is a top view of the atomic force microscope photograph of the silver array produced in the Example. It is an enlarged view of the atomic force microscope photograph of the silver array produced in the Example. It is a three-dimensional solid view of the atomic force microscope photograph of the silver array produced in the Example. It is a graph which shows the surface enhancement Raman spectrum and (B) background of the (A) blinking part in an Example. It is a graph which shows the frequency of blinking on the part (A) of the silver array in an Example, and the thin film (B) of a 50 nm film thickness.

Explanation of symbols

Forming part of a single particle layer film of 1 space Sa <br/> 2 suspension 3 glass substrate 4 a glass substrate 5 nanoparticles Nanoparticles

Claims (4)

In the method for producing a substrate having a metal nano-triangular prism structure array formed by forming a close-packed monolayer film of nanoparticles on a substrate, vacuum-depositing a metal on the substrate, and washing away the nanoparticles. The formation of a monolayer film in which particles are two-dimensionally arranged uses two glass substrates with different affinity for the nanoparticles, and one substrate with high affinity is placed horizontally and the nanoparticles on the surface. The other substrate having a lower affinity is placed on the spacer, and one end of the substrate is leaned against the spacer, placed at an angle, and dried in an apparatus in which the humidity is controlled to 60% or more. A method for producing a substrate having a metal nano-triangular prism structure array, characterized in that   The method for producing a substrate according to claim 1, wherein both of the two substrates are made of glass, and the hydrophilic strength of each glass surface is different.   A method for analyzing the state of a single molecule comprising measuring a surface-enhanced Raman scattering spectrum and its blinking in an array of metal nanotriangular prism structures using the substrate prepared by the method according to claim 1 or 2. A substrate used in a method for analyzing the state of a single molecule, which is prepared by the method of claim 1 or 2 and which measures a surface-enhanced Raman scattering spectrum and its blinking in an array of metal nanotriangular prism structures.

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