TWI480537B - Substrate for raman spectroscopy and method of manufacturing the same - Google Patents

Description

Substrate for Raman spectroscopy and manufacturing method thereof

The present invention relates to a substrate for Raman spectroscopy and a method for producing the same, wherein the substrate for the Raman spectroscopy technique is especially a substrate for surface enhanced Raman spectroscopy.

Raman spectroscopy enables rapid detection of a wide range of samples, including the need for additional complex sample preparation, non-destructive detection, and direct detection of all types of gaseous, liquid, and solid molecules, including: Protein, nucleic acid, nuclear protein, cell membrane or lipid.

Raman spectroscopy identifies the structure of a molecule through the interaction between molecular and photon energy, making it a fast and easy method of detection. Raman spectroscopy also includes a surface-enhanced Raman spectroscopy (SERS) technique. When inert metal nanoparticles close to each other are exposed to appropriate incident light, the surface of the metal nanoparticles induces surface plasmon resonance. Coupling with the plasma around the metal nanoparticles, the collective oscillation of the surface plasmons significantly enhances the cross-sectional area of the absorption/scattering of the nanoparticles, and at the same time increases the electric field at the surface of the local metal nanoparticles. Since the intensity of the Raman spectral signal is proportional to the electric field strength, the stronger the electric field generated on the surface of the metal nanoparticle, the stronger the intensity of the Raman signal generated, thereby improving the detection sensitivity of the Raman spectroscopy technique, thereby enabling Surface-enhanced Raman spectroscopy has become an application technology with extremely high potential, which can quickly detect various trace samples and apply it to biomolecule identification, drug detection, medical diagnosis, analysis and identification.

However, when performing the Raman spectroscopy test, the nanoparticles in the substrate may be damaged by the environment or the analyte. Therefore, in order to overcome the above problems, the present invention provides a substrate for Raman spectroscopy which protects the nanoparticles from damage and provides recyclability of the substrate.

The main object of the present invention is to provide a substrate for Raman spectroscopy and a method for fabricating the same, which can enhance the Raman signal intensity of an analyte (such as a biomolecule or a drug) and enhance the detection capability of Raman spectroscopy.

Another object of the present invention is to provide a method for producing a substrate for Raman spectroscopy, which can protect a metal nanoparticle in a substrate for Raman spectroscopy from environmental or analyte contamination or damage. The substrate can have the advantage of being reused and a substrate for chemically inert and biocompatible Raman spectroscopy can be made.

In order to achieve the above object, the present invention provides a method for producing a substrate for Raman spectroscopy, comprising the steps of: (A) providing a substrate; (B) forming a plurality of metal nanoparticles; and (C) providing a layer Or a graphene layer on the metal nanoparticles, and surface-treating the graphene layers to form one or more surface-treated graphene layers.

Wherein, in step (B), the synthetic metal nanoparticle can be directly disposed; or, a metal film can be formed on the substrate, and the metal film is processed to form metal nanoparticles disposed on the substrate. Herein, the method of treating the metal thin film may be an annealing method to convert the metal thin film into metal nano particles.

Further, in the step (C), the surface treatment of the graphene layer is preferably performed by exposing the graphene layers to a hydrogen-containing atom, an oxygen-containing atom, a hydrogen-containing radical, or an oxygen-containing radical. The surface treatment step is completed in the environment; or, preferably, the surface of the graphene layer is treated by plasma, and more preferably, the surface of the graphene layer is treated by microwave plasma to form an oxide graphene layer or a hydrogenated graphene layer. (hydrogenated graphene).

In addition, in order to achieve the above object, the present invention also provides a substrate for Raman spectroscopy, comprising: a substrate; a plurality of metal nanoparticles, wherein the metal nanoparticles are disposed on the substrate, and the metals The nanoparticles are disposed at a distance from each other; and one or more surface-treated graphene layers are disposed above the metal nanoparticles.

The material of the metal nanoparticle may be silver, gold, copper, nickel, cobalt, iron, platinum, palladium or aluminum, preferably silver, gold, copper, nickel or platinum, more preferably silver, gold or platinum. The metal nanoparticles may have a particle diameter of from 2 nm to 400 nm, preferably from 5 nm to 400 nm, more preferably from 40 nm to 125 nm. Further, the metal nanoparticles may be disposed between 5 nm and 400 nm, preferably 5 nm to 150 nm, more preferably 20 nm to 150 nm. Further, the surface-treated single-layer graphene layer has a thickness of about 0.4 nm to 3 nm, preferably 0.5 nm to 2 nm. Wherein, a plurality of layers of the treated graphene layer may be additionally disposed on the metal nanoparticles to increase the thickness. When detecting the Raman spectrum of an analyte, the analyte can be applied to the graphene layer of the substrate for Raman spectroscopy, and the concentration of the analyte is about 1 nM to 1 M, preferably 1 nM to 0.01. M, more preferably from 1 μM to 0.01 M, still more preferably from 10 μM to 0.01 M.

The substrate for Raman spectroscopy provided by the invention can be applied to various Raman spectroscopy techniques, such as surface enhanced Raman spectroscopy (SERS) and surface enhanced resonance Raman spectroscopy. , SERRS), coherent anti-Stokes Raman Spectroscopy.

The invention further provides a method for determining Raman spectroscopy, comprising the following steps: (A) providing a substrate for a Raman spectroscopy technique, the substrate for the Raman spectroscopy technique comprising: a substrate; a plurality of metal nanoparticles a particle, the metal nanoparticle layer is disposed on the substrate, and the metal nanoparticles are disposed at a distance from each other; and one or more graphene layers are disposed on the metal naphthalene layer Above the rice particles; (B) placing an analyte above the substrate for the Raman spectroscopy technique; (C) exciting a laser beam to generate a Raman signal; (D) detecting a Raman signal, The Raman signal is detected by a Raman detector.

Wherein, the laser light is irradiated to the substrate for the Raman spectroscopy technique to generate a local electric field between the plurality of metal nanoparticles, and a Raman signal is generated by the coupling of the electric field between the analyte and the metal nanoparticle.

The graphene layer including the plasma hydrogenation or oxidation treatment can form a graphene oxide or a hydrogenated graphene layer, and the resistivity of the graphene can be improved by plasma treatment, and the influence of different microwave plasma treatment time on the graphene is not Do the same. The invention provides a microwave plasma treated graphene layer on the substrate, which can make a stronger local electric field coupling between the analyte on the substrate and the metal nanoparticle, thereby obtaining strong Raman scattering intensity. Improve the detection capability of Raman spectroscopy. Therefore, the present invention provides a simple method for improving the intensity of Raman signals by using a thin layer of oxidized or hydrogenated graphene on the metal nanoparticles to substantially increase the signal intensity of the analyte in the Raman spectrum. Mann spectroscopy is more widely used to detect trace amounts of molecules.

In addition, in the substrate for Raman spectroscopy provided by the present invention, the graphene layer can also serve as a protective layer of the substrate, and the analyte and the metal nanoparticle are separated by an extremely thin graphene layer to protect the metal naphthalene. Rice particles are protected from environmental or analyte contamination or damage, reducing the cost of detecting various molecules using Raman spectroscopy. Therefore, the present invention provides that Raman spectroscopy technology can be widely applied to the detection of biomolecules, and the substrate for Raman spectroscopy technology can be reused and can obtain the advantages of stable Raman signals, thereby providing a more environmentally friendly Economical substrate.

<First Embodiment>

Please refer to FIG. 1A to FIG. 1D , which are flowcharts of a method for manufacturing a substrate for Raman spectroscopy provided by the present invention. The manufacturing method includes the following steps: First, an oxidized germanium wafer substrate 11 is provided. The substrate 11 has a ruthenium substrate 111 and a ruthenium dioxide layer 112 disposed above the ruthenium substrate 111, as shown in FIG. 1A.

Next, a silver metal film 121 is formed on the substrate 11 by a thermal evaporation method in a high vacuum environment, and the thickness of the silver metal film 121 is 5 nm as shown in FIG. 1B.

Thereafter, the silver metal thin film 121 is processed by a thermal annealing method, and the silver metal thin film 121 is separated to form a plurality of metal nanoparticles 122. Wherein, the nano silver particles are uniformly distributed on the germanium wafer substrate 11, the distance between the nano silver particles is about 20 nm to 120 nm, and the diameter of the nano silver particles is about 20 nm to 120 nm, such as Figure 1C shows.

Further, a single layer of graphene layer (not shown) was prepared by electrolyzing a mixed gas of methane and hydrogen on copper platinum by low-pressure thermal chemical vapor deposition. Among them, the gas pressure is about 0.1 to 100 torr, and the reaction temperature is about 900 to 1035 °C. After the graphene is grown on the copper foil, the characteristic peaks D-band, G-band and 2D-band of the synthesized single-layer graphene can be examined by Raman spectroscopy. Band) to confirm the graphene structure. The synthesized graphene can be removed from the copper foil by wet chemical etching, and the graphene layer 13 is transferred over the metal nanoparticles 122 disposed on the substrate 11. In addition, the present invention provides a method for manufacturing a substrate for Raman spectroscopy, which can be further treated by microwave plasma treatment, with an oxygen-containing ambient air of 1-5 torr, a microwave power of 400 W, and a microwave frequency. At 2.45 GHz, the reaction is continued for 2 to 5 seconds, whereby the synthesized graphene layer 13 is oxidized to obtain an oxidized graphene layer 13, and the substrate 1 for Raman spectroscopy is completed, as shown in FIG. 1D. . Here, the microwave plasma treatment of graphene does not require an additional heat treatment step.

Accordingly, referring to FIG. 2, a substrate 1 for Raman spectroscopy technology obtained in the first embodiment of the present invention has a substrate comprising: a germanium dioxide substrate 11 having a germanium substrate 111 and a ruthenium dioxide layer 112 located above the ruthenium substrate 111; a plurality of metal nanoparticles 122 disposed on the substrate 11 and disposed between the metal nanoparticles 122 at a distance from each other; One or more oxidized graphene layers 13 are disposed above the metal nanoparticles 122 to form a substrate 1 for Raman spectroscopy.

<Second embodiment>

The substrate for the Raman spectroscopy technique of the second embodiment of the present invention is produced by the method of the first embodiment, and differs only in the method of how to treat the graphene layer by microwave plasma.

In the second embodiment, after the graphene is grown on the copper foil, and the graphene layer 13 is transferred to the metal nanoparticle 122 disposed on the substrate 11, the hydrogen gas is 1-5 torr. The microwave power is 500 W, the microwave frequency is 2.45 GHz, and the graphene layer is treated by microwave plasma for 5 to 10 seconds, thereby hydrogenating the synthesized graphene layer to obtain a hydrogenated graphene layer. Thereafter, as in the method described in the first embodiment, a substrate for a Raman spectroscopy technique having a hydrogenated graphene layer was obtained.

<Third embodiment>

In the third embodiment of the present invention, a substrate for a Raman spectroscopy technique having a hydrogenated graphene layer was produced by the method of the second embodiment, except that the experimental parameters of the graphene layer were treated by microwave plasma.

In this embodiment, the hydrogen gas is 1 torr, the microwave power is 200 W, the microwave frequency is 2.45 GHz, the plasma ball system is disposed at a distance of about 10 cm from the graphene layer, and the plasma is separated by a metal film containing a plurality of holes. The ball and the graphene layer are continuously reacted for 5 to 60 minutes, whereby the graphene layer is slowly hydrogenated to obtain a hydrogenated graphene layer. Finally, as in the method described in the first embodiment, a substrate for a Raman spectroscopy technique having a hydrogenated graphene layer is obtained.

<Fourth embodiment>

Referring to FIG. 3, in the fourth embodiment of the present invention, the analyte is placed above the graphene layer 13 of the substrate 1 for Raman spectroscopy for measurement of Raman spectroscopy.

First, the substrate 1 for the Raman spectroscopy technique fabricated in the first to third embodiments can be used.

Thereafter, the substrate 1 for this Raman spectroscopy technique is immersed in a 0.001 M to 0.01 M adenine solution for 30 minutes to 1 hour, and washed with deionized water to make the analyte 14 (ie, adenine). The molecule is disposed above the graphene layer 13 of the substrate 1 for Raman spectroscopy.

Accordingly, the substrate for the Raman spectroscopy technique comprises: a ruthenium dioxide substrate 11 having a ruthenium substrate 111 and a ruthenium dioxide layer 112 above the ruthenium substrate 111; and a plurality of metal nanoparticles 122, the metal The nanoparticle 122 is disposed on the substrate 11 and disposed between the metal nanoparticles 122 at a distance from each other; and a graphene layer 13 disposed above the metal nanoparticles 122 . Further, above the substrate 1 for Raman spectroscopy, a plurality of analytes 14 (adenine molecules) are disposed above the graphene layer 13 of the substrate 1 (as shown in FIG. 3).

<Comparative example>

In the comparative example of the present invention, a substrate for Raman spectroscopy is produced similarly to the method of the first embodiment of the present invention, except that the graphene layer is a single layer of graphite which has not been treated by other microwave plasma. Ene layer.

<First Test Example>

The first test example measures the resistance value of the graphene layer in the substrate of the third embodiment and the comparative example. In this test example, the nano silver particles in the substrate are arranged at intervals of 100 nm, and two gold electrodes are deposited at the ends of the 5 mm × 5 mm graphene layers to measure the graphite of the third embodiment and the comparative examples. The resistance value of the olefin layer.

As shown in FIG. 4, the graphene layer (comparative example) which has not been subjected to microwave plasma treatment has a resistance value of 2.5 kΩ, and the microwave plasma treatment time is extended for 60 minutes after being treated with microwave microwave plasma for 60 minutes. The resistance of the hydrogenated graphene layer will increase to 50 kΩ. The experimental results show that the resistance of the graphene layer by microwave plasma can increase the electrical resistance by more than 20 times.

Therefore, by setting a graphene layer treated by microwave plasma on top of the metal nanoparticle, a higher electric field can be formed on the graphene layer, so that more analytes are excited to achieve Raman enhancement of the analyte. Signal strength efficiency.

<Second test example>

The method for determining the Raman spectroscopy technique of the present invention comprises the following steps: First, the substrate for the Raman spectroscopy technique prepared by the first embodiment to the second embodiment is used as the surface enhanced Raman spectroscopy technique in the test example. The substrate used.

Thereafter, a laser light having a wavelength of 532 nm is excited, and the laser light is irradiated to the substrate for Raman spectroscopy to generate an electric field between the metal nanoparticles to generate a Raman signal.

Finally, the generated Raman signal is detected by a Raman detector and converted into a surface enhanced Raman spectrum by calculation.

Referring to FIG. 5, it is a surface-enhanced Raman spectrum of a substrate for Raman spectroscopy, in which a graphene layer is subjected to microwave plasma treatment of a single layer of graphene oxide layer (a), passing through a microwave. The plasma treated single layer hydrogenated graphene layer (b) and the single layer graphene layer (c) which has not been subjected to microwave plasma treatment.

The characteristic peaks of graphene in the surface-enhanced Raman spectrum of the comparative example (the graphene layer not subjected to microwave plasma treatment) are D-band, G-band and 2D-, respectively. With 2D-band, the D-band is weak, the G-band has a strong signal, and the 2D-band has a symmetrical signal (as shown in Figure 5), showing the unprocessed single-layer graphene. The layer has good crystallinity.

In the surface-enhanced Raman spectrum of the substrate in the first embodiment (graphene oxide layer), the signal intensity of 2D-band is significantly weaker than that of D-band and G-band (as shown in FIG. 5). The result of the spectroscopy is the same as the Raman spectrum of the conventional graphene layer oxide, showing the substrate in the first embodiment, and the graphene layer is actually treated by microwave plasma to achieve a partial graphene oxide layer effect. .

In the surface-enhanced Raman spectrum of the substrate in the second embodiment (hydrogenated graphene layer), the signal intensity of D-band is significantly stronger than that of the untreated graphene layer, and the signal intensity of 2D-band is The weakest of the three signals (as shown in Figure 5), the result of this Raman spectrum is the same as the Raman spectrum of a conventional graphane (hydrogenated graphene layer), showing the second embodiment The substrate, the graphene layer does have the effect of hydrogenating the graphene layer by microwave plasma treatment.

<Third test example>

The third test example of the present invention is a surface-enhanced Raman spectroscopy experiment in which an analyte (adenine molecule) is placed on a substrate using an experimental method as in the second test example.

First, an analyte (adenine molecule) was separately provided to the substrate for the Raman spectroscopy technique produced in the first embodiment and the second embodiment.

Thereafter, a laser light having a wavelength of 532 nm is excited, and the laser light is irradiated to the substrate for Raman spectroscopy to generate an electric field between the metal nanoparticles. Next, an electric field is generated by the electric field between the adenine molecule and the metal nanoparticle to generate a Raman signal. Herein, the substrate for Raman spectroscopy provided by the present invention can increase the Raman signal intensity of the analyte. Finally, the generated Raman signal is detected by a Raman detector and converted into a surface enhanced Raman spectrum by calculation.

Referring to Figure 6, a comparison of Raman spectra obtained by detecting adenine molecules using a substrate using different Raman spectroscopy techniques. Among the selected substrates, the graphene layer is a single layer of graphene oxide layer (a) treated with microwave plasma, a single layer of hydrogenated graphene layer (b) and a single layer of graphene without microwave plasma treatment. Layer (c). In the surface-enhanced Raman spectrum, a signal of 734 cm ^-1 Raman shift is observed for adenine molecules (analytes). Comparing the substrate of the untreated graphene layer with the substrate of the graphene layer subjected to microwave plasma treatment, the signal of adenine molecules cannot be observed in the substrate of the monolayer graphene layer which is not subjected to microwave plasma treatment; However, in the Raman spectrum of the substrate of the first embodiment (the graphene oxide layer) and the second embodiment (the hydrogenated graphene layer), the signal of the adenine molecule was clearly observed.

It can be seen that the microwave-plasma treatment method can localize the π-electron in the graphene, and reduce the conductivity of the graphene. The microwave-treated graphene is advantageous for use as a good ultra-thin protective layer in a substrate for Raman spectroscopy, and enables a substrate having a surface-treated graphene layer to enhance metal nanoparticle detection. The sensitivity of the analyte gives a strong analyte Raman signal intensity. Therefore, the substrate for Raman spectroscopy provided by the present invention can enhance the ability of Raman spectroscopy to detect analytes and effectively increase the Raman signal intensity of the analyte.

<Fourth test example>

The fourth test example compares Raman spectra obtained from substrates used in different Raman spectroscopy techniques. In this test example, the hydrogenated graphene layer (third embodiment) treated with microwave plasma for 5 minutes (a) or 60 minutes (b) and the single-layer graphene layer without microwave plasma treatment were respectively (Comparative Example) The Raman spectrum signal was compared between the metal nanoparticles and the laser excitation light having a wavelength of 633 nm to irradiate the substrate.

Referring to FIG. 7, the surface-enhanced Raman spectrum of the comparative example has obvious signals in D-band, G-band and 2D-band; in the hydrogenated graphene layer treated by microwave plasma for 5 minutes, D-band The intensity of the signal is significantly stronger, and the signal intensity of the 2D-band is the weakest of the three signals; while the hydrogenated graphene layer is treated by microwave plasma for 60 minutes, the graphene layer is not completely hydrogenated to graphane. Instead, a hydrogenated graphene layer having a mixture of graphene and graphane is formed.

<Fifth Test Example>

The fifth test example compares Raman spectra obtained by detecting adenine molecules using a substrate using different Raman spectroscopy techniques. In the test examples, the substrate used was a substrate (a) having only a graphene layer (non-nano silver particles) on the substrate, and only nano silver particles on the substrate (no graphene above the nano silver particles) a substrate (b) of the layer), a substrate (c) (Comparative Example) in which a graphene layer not subjected to microwave plasma treatment is disposed above the nano silver particles on the substrate, and a microwave plasma treatment with hydrogen for 60 minutes Substrate (d) for the spectroscopy technique (third embodiment). The Raman shift is 721 cm ^-1 and 732 cm ^-1 is the Raman signal of the adenine molecule. As shown in Fig. 8, when a substrate having only a graphene layer (non-nano silver particles) on the substrate is used, the Raman signal of any adenine molecule cannot be detected; when the substrate is used, only nano silver particles are used ( A weak adenine molecular Raman signal was observed on the substrate without the graphene layer above the nano silver particles; however, the hydrogenated graphene layer treated by microwave plasma for 60 minutes was used as the base for Raman spectroscopy. In the case of the material, a strong analyte (adenine molecule) Raman signal was observed, indicating that the graphene layer was treated by microwave plasma for 60 minutes, and the resistance value was increased to 50 kΩ. The reason why the resistance value is increased is that the use of the hydrogenated graphene layer as a protective layer for the substrate for Raman spectroscopy enables a stronger local electric field coupling effect between the analyte disposed on the substrate and the metal nanoparticle. , to obtain strong Raman scattering intensity, improve the detection ability of Raman spectroscopy technology.

It can be seen that the substrate for Raman spectroscopy manufactured by the present invention can effectively enhance the Raman signal of the analyte by treating the graphene layer through microwave plasma, and at the same time, by chemical inertness and biocompatibility of graphene. The substrate of the present invention is more suitable as a substrate for surface enhanced Raman spectroscopy.

The above-mentioned embodiments are merely examples for convenience of description, and the scope of the claims is intended to be limited to the above embodiments.

1. . . Substrate for Raman spectroscopy

11. . . Oxidized tantalum substrate

111. . .矽 substrate

112. . . Ceria layer

121. . . Metal film

122. . . Metal nanoparticles

13. . . Graphene layer

14. . . Analyte

1A to 1D are flow charts showing a method of manufacturing a substrate for Raman spectroscopy of the present invention.

Figure 2 is a structural view of a substrate for Raman spectroscopy of the present invention.

Fig. 3 is a structural view showing the arrangement of an analyte above a substrate for Raman spectroscopy in a fourth embodiment of the present invention.

Figure 4 is a graph showing the resistance-time relationship of the resistance of the graphene layer to different microwave plasma treatment times.

Fig. 5 is a second test example of the present invention which is a Raman spectrum obtained by comparing substrates used in different Raman spectroscopy techniques.

Fig. 6 is a third test example of the present invention, which is a Raman spectrum obtained by comparing adenine molecules obtained using substrates of different Raman spectroscopy techniques.

Fig. 7 is a fourth test example of the present invention, which is a Raman spectrum obtained by comparing substrates used in different Raman spectroscopy techniques.

Fig. 8 is a fifth test example of the present invention, which is a Raman spectrum obtained by comparing adenine molecules obtained using substrates of different Raman spectroscopy techniques.

1. . . Substrate for Raman spectroscopy

11. . . Oxidized tantalum substrate

111. . .矽 substrate

112. . . Ceria layer

121. . . Metal film

122. . . Metal nanoparticles

13. . . Graphene layer

Claims (16)

A method for manufacturing a substrate for Raman spectroscopy comprises the steps of: (A) providing a substrate; (B) forming a metal film on the substrate, and processing the metal film to form a plurality of metal nanoparticles. And (C) providing one or more graphene layers on the metal nanoparticles, and surface treating the graphene layers to form one or more surface-treated graphene layers, wherein the surface treatment The graphene layer is a graphene oxide layer or a hydrogenated graphene layer. The method for producing a substrate for Raman spectroscopy according to claim 1, wherein in the step (C), the graphene layer is exposed to a hydrogen atom, an oxygen atom, The graphene layers are surface treated in the presence of a hydrogen radical or an oxygen radical. The method for producing a substrate for Raman spectroscopy according to claim 1, wherein in the step (C), the graphene layers are surface-treated by plasma treatment. The method for producing a substrate for Raman spectroscopy according to the first aspect of the invention, wherein in the step (B), the metal thin film is treated by an annealing method to form the metal nanoparticles. The method for producing a substrate for Raman spectroscopy according to the first aspect of the invention, wherein the metal nanoparticles have a particle diameter of 5 nm to 400 nm. The method for producing a substrate for Raman spectroscopy according to claim 1, wherein the surface-treated graphene layer has a single layer thickness of 0.4 nm to 3 nm. The method for producing a substrate for Raman spectroscopy according to claim 1, wherein the material of the metal nanoparticle is silver, gold, copper, nickel, cobalt, iron, platinum, palladium or aluminum. . A substrate for Raman spectroscopy, comprising: a substrate; a plurality of metal nanoparticles, wherein the metal nanoparticles are disposed on the substrate, and the metal nanoparticles are disposed at a distance from each other; One or more surface-treated graphene layers, the surface-treated graphene layer being disposed above the metal nano-particles, wherein the surface-treated graphene layer is a graphene oxide layer or A hydrogenated graphene layer. The substrate for Raman spectroscopy according to claim 8, wherein the surface-treated graphene layer is formed by plasma treatment. The substrate for Raman spectroscopy according to claim 8, wherein the metal nanoparticles have a particle diameter of 5 nm to 400 nm. The substrate for Raman spectroscopy according to claim 8, wherein the metal nanoparticle is made of silver, gold, copper, nickel, cobalt, iron, platinum, palladium or aluminum. The substrate for Raman spectroscopy according to claim 8, wherein the metal nanoparticles are separated by 5 nm to 150 nm. The substrate for Raman spectroscopy according to claim 8, wherein the surface-treated graphene layer has a single layer thickness of 0.4 nm to 3 nm. The substrate for Raman spectroscopy according to claim 8, wherein the substrate for the Raman spectroscopy is formed by: providing a substrate; forming a plurality of metal naphthalenes on the substrate And arranging one or more graphene layers on the metal nanoparticles, and surface treating the graphene layers to form one or more surface-treated graphene layers. The substrate of the Raman spectroscopy technique of claim 8, wherein the method further comprises disposing an analyte over the graphene layer of the substrate for the Raman spectroscopy. The substrate of the Raman spectroscopy technique of claim 15, wherein the analyte has a concentration of from 1 nM to 1 M.