TWI657166B - Handheld raman detection test paper and manufacture method and use thereof - Google Patents

Abstract

The invention provides a method for manufacturing a portable Raman optical test strip, which comprises the following steps: (a) providing a substrate which is a water absorbing material having a micron or submicron fiber structure; (b) immersing the substrate to a reduction In the agent, the reducing agent is attached to the micro or sub-micron fiber structure; and (c) the metal salt solution is dropped onto the substrate to react the metal salt solution with the reducing agent, and the nano- or sub-micron fiber structure is grown on the nano- or sub-micron fiber structure. Metal particles. The invention also provides a portable micro-Raman optical test strip and a method for detecting a test paper by Raman optical.

Description

Portable Raman optical test strip, preparation method and use method thereof

The invention relates to an optical detecting test paper, in particular to a portable micro-Raman optical testing test paper and a preparation method and a using method thereof.

Raman spectroscopy is a Raman scattering spectrum of a molecule that corresponds to the structure of the molecule itself, so the Raman spectra of different molecules have different characteristics. If the Raman spectrum of an unknown substance is compared with the Raman spectrum of a known substance, the composition of the qualitative unknown substance can be analyzed. However, the spontaneous Raman scattering of the molecule is very weak, so the resulting Raman spectrum signal is weak, leading to analytical difficulties.

In recent years, due to the development of Surface-Enhanced Raman Scattering (SERS), the development and application of surface-enhanced Raman spectroscopy in scientific research and testing have been enhanced. Surface-enhanced Raman spectroscopy is to adsorb the molecules to be tested on the metal surface of a special structure, so that the Raman scattering signal of the molecule to be tested is enhanced, so that the Raman scattering signal is sufficient to detect the extremely low concentration of the molecule to be tested, thus extending the surface enhancement. The range of applications of Raman spectroscopy. For example, micro-detection analysis, cancer cell calibration, and SERS technology to detect molecular information in living organisms.

The existing surface-enhanced Raman spectroscopy substrates are generally satisfactory, but are not satisfactory in various aspects. For example, in the preparation of substrates, expensive instruments are required, resulting in high cost, and thus there is still room for improvement.

The invention provides a method for manufacturing a portable Raman optical test strip comprising the steps of: (a) providing a substrate which is a water absorbing material having a micron or submicron fiber structure; and (b) immersing the substrate to In the reducing agent, the reducing agent is attached to the micron or sub-micron fiber structure; and (c) the metal salt solution is dropped onto the substrate to react the metal salt solution with the reducing agent, and the nano- or sub-micron fiber structure is grown on the nano- or sub-micron fiber structure. Metal particles.

The present invention further provides a portable micro-Raman optical test paper comprising: a substrate having a water-absorbing material of a micron or sub-micron fiber structure; and a nanometer or a distribution of nano metal particles on the surface and inside of the substrate Sub-micron fiber structure.

The invention further provides a method for using a portable micro-Raman optical test strip, comprising the steps of: dropping the molecule to be tested into the portable Raman optical test strip of the invention, wherein the molecule to be tested is micrometer or sub-micrometer of the test strip Adsorption of fiber structure; surface-enhanced Raman spectroscopy of test paper; surface-enhanced Raman spectroscopy to determine the type of molecule to be tested.

The above described objects, features, and advantages of the invention will be apparent from the description of the appended claims.

101, 102, 103, 104, 105 ‧ ‧ steps

201‧‧‧Fiber structure

202‧‧‧3D construction

203‧‧‧solution

204‧‧‧Biological macromolecules

205‧‧‧ compounds

Lines a, b, c‧‧

Fig. 1 is a flow chart showing an embodiment of a portable micro-Raman optical test strip of the present invention.

Figure 2 is an implementation of the portable micro-Raman optical test strip of the present invention. A schematic diagram of the use of the example.

Fig. 3 is an image of a scanning electron microscope of an embodiment of the portable micro-Raman optical test strip of the present invention.

Fig. 4 is a comparison diagram of the nano gold particles produced by the embodiment of the portable micro-Raman optical test strip of the present invention and the test paper of a comparative example.

Fig. 5 is a comparison diagram of another embodiment of the portable micro-Raman optical test paper of the present invention and nano-gold particles formed on the fiber surface of another comparative test paper.

Fig. 6 is a view showing the results of Raman spectroscopy analysis of different kinds of molecules to be tested using another embodiment of the portable micro-Raman optical test strip of the present invention and a test paper of still another comparative example.

Fig. 7 is a view showing the results of Raman spectroscopy analysis of molecules of different concentrations of the test molecules using the test paper of another embodiment of the portable micro-Raman optical test strip of the present invention.

The existing surface-enhanced Raman scattering substrate is required to be sputtered to the surface of the hard substrate by vacuum sputtering. This method is not only expensive, but also expensive, resulting in high cost.

In view of the above, the method for manufacturing a portable micro-Raman optical test strip provided by the present invention uses only the steps of soaking, dripping, etc., and does not require an additional instrument, and can be fabricated under normal temperature and pressure, thereby greatly reducing cost.

Please refer to FIG. 1. FIG. 1 is a flow chart showing a method of manufacturing the portable micro-Raman optical test strip of the present invention. First, step 101 (providing the substrate), followed by step 102 (soaking the substrate to the reducing agent), and then performing the steps. 104 (drop the metal salt solution onto the substrate soaked with the reducing agent). After step 102 (substrate soaking of the reducing agent), or after dropping the metal salt solution, step 103 (drying) or step 105 (drying) may be included. Hereinafter, a method of manufacturing the portable micro-Raman optical test strip of the present invention will be described in detail.

In the step 101 (providing a substrate), the substrate used in the present invention is a water absorbing material having a fiber structure of micron (1 to 1000 μm) or submicron (0.01 μm or more, less than 1 μm). The substrate can be used with a variety of materials having micro or sub-micron fiber structures, such as paper, sponge, cotton swabs, or combinations thereof, wherein the paper includes filter paper, white paper, or newspapers. The thickness of the substrate is not particularly limited and may generally be 0.05 to 1.5 mm, for example, 0.12 to 0.8 mm. The test paper of the present invention can produce a test paper having nano metal particles on both the surface and the inside of the test paper by using a water absorbing material having a micron or submicron fiber structure as a substrate, and this portion will be described in detail later.

In addition, the present invention uses a substrate having a micron or sub-micron fiber structure as compared with a conventional rigid substrate such as an alumina substrate, a tantalum substrate, or a glass substrate. Therefore, the test paper of the present invention is flexible and is being pulled. Mann spectral analysis is not limited by the curvature and shape of the analyzed area. Furthermore, the conventional alumina substrate, tantalum substrate, glass substrate, and the like are non-combustible substrates, and the portable micro-Raman optical test strip of the present invention is made of a flammable material, so it has the advantage of being disposable and environmentally friendly. Sex.

Next, proceed to step 102 (soak the substrate to a reducing agent). The reducing agent used in the production method of the present invention is a natural reducing agent. The reducing agent is natural tea polyphenol, curcumin, citric acid, vitamin c and the like. The reducing agent can be dissolved in a solvent, and various solvents such as water or alcohol can be used as needed. Reducing agent concentration It is between 1 and 50 mM, for example 10 mM.

It should be noted that the method of the present invention requires only a single type of natural reducing agent. In general, two or more reducing agents must be used in the manufacture of Raman test strips in order to obtain a sufficient reduction effect. The present invention increases the reaction surface area ratio by using micro or sub-micron fibers by using a substrate having a micron or sub-micron fiber structure, so that even if only one reducing agent is added, a sufficient reduction effect can be achieved.

The invention adopts a method of immersing a substrate into a reducing agent, so that the reducing agent can be impregnated into the micro or sub-micron fiber structure inside the substrate, the area of contact of the reducing agent with the micron or sub-micron fiber is increased, and the distribution of the reducing agent is uniform. Although the reducing agent can be added to the substrate by using the spraying method, the spraying can only reach the surface of the substrate compared with the immersion method, and the substrate cannot have a reducing agent on the surface and the inside, and the spraying agent is easy to have a reducing agent on the substrate. The problem of uneven surface distribution.

The embodiment of the immersion is not particularly limited as long as the substrate can be immersed in the reducing agent. For example, a container may be used to contain a reducing agent, and then the substrate may be immersed in a container; or the substrate may be bubbled in a solvent, followed by addition of a reducing agent and stirring. The soaking time can be adjusted according to actual needs, for example, can be adjusted according to the type or concentration of the reducing agent, and the time can be 10 seconds to 10 minutes, for example, 5 minutes. The soaking temperature can also be adjusted according to the type or concentration of the reducing agent, and can be 4 to 100 ° C, for example, 50 ° C.

After soaking, place the substrate on a lens paper or other absorbent medium or other means to remove excess reducing agent. Step 103 (drying) can then be performed as needed. Drying can be maintained at 40~80 °C for 1~10 minutes. In one embodiment, drying is maintained at 50 ° C for 5 minutes, or at room temperature for natural drying in another embodiment In the middle, the drying is not performed, and the test paper prepared in a wet state also has a Raman amplification effect.

Next, step 104 is performed (a metal salt solution is dropped on the substrate). The metal salt solution can be a chloroauric acid solution, a silver nitrate solution, or a combination thereof. When a metal salt solution is used alone, a single metal particle can be grown on the micron or submicron fiber structure, and if two different metal salt solutions are dropped or a mixed solution of the two metal salts is dropped, it can be in micrometer or submicron. The alloy metal particles are grown on the fiber structure. For example, gold nitrate alloy nanoparticles can be produced by dropping a silver nitrate solution before dropping the chloroauric acid solution, and the desired alloy ratio can be produced by adjusting the ratio of the metal salt solution.

The concentration of the chloroauric acid solution used in the present invention is 1 to 10 mM, for example, 5 mM. The concentration of the silver nitrate solution is 0.001 to 15 mM, for example, 0.1 mM. The pH of the metal salt solution used in the present invention may be acidic or neutral, for example, may be pH 2-7, and in one embodiment, the pH is 2.35.

The instilled metal salt solution is subjected to a spontaneous redox reaction with a reducing agent on the micron or submicron fiber to directly grow the nano metal particles on the surface of the micron or submicron fiber. In the conventional Raman test paper manufacturing method, it is necessary to first synthesize nano metal particles, and then apply a solution containing nano metal particles to the substrate. Therefore, a solution such as a protective agent or a dispersant is added to the solution to prevent the solution from being dissolved. The nano metal particles produce agglomeration. However, these additives themselves also have Raman scattering, which affects the interpretation of Raman spectroscopy.

Since the nano metal particles of the present invention directly grow nano metal particles on the micron or submicron fibers by the redox reaction of the metal salt solution, no additional additives are required, and the accuracy of the Raman spectrum interpretation can be increased.

In addition, since the substrate used in the present invention is a water absorbing material and is immersed in a reducing agent, the reducing agent and the metal salt solution can penetrate into the micro or sub-micron fiber structure inside the substrate, and the micro or sub-micron inside. Since the nano metal particles are grown on the fibers, the test paper of the present invention can be used to produce a test paper having nano metal particles formed on the surface and inside of the test paper. More specifically, the test strip of the present invention has nano metal particles ranging from at least 300 nm to 10 mm from the surface of the substrate to the inside of the substrate. In one embodiment, there are nano metal particles from the surface of the substrate to the back side of the substrate (0.2 mm).

Next, the substrate into which the metal salt solution is dropped is subjected to step 105 (drying). Drying can be maintained at 40~80 °C for 1~10 minutes, or step 105 (drying). In one embodiment, the drying is maintained at 50 ° C for 5 minutes. In addition, the humidity of the drying vessel is not limited, and the magnifying test paper can be synthesized from high humidity to low humidity, for example, the humidity can be 25%.

The above steps 102-105 are one-time flow. In order to achieve the purpose of growing more nano metal particles on the micron or sub-micrometer fibers, the steps 102 to 105 may be repeated a plurality of times, for example, 2 to 10 times. When the steps 102 to 105 are repeated a plurality of times, whether or not the drying steps 103 and 105 are performed may be selected as needed.

According to the above steps, the micro-Raman optical test strip of the present invention can be produced for surface-enhanced Raman spectroscopy. The detection method using the test paper of the invention is also very simple, and only the solution containing the molecule to be tested is dropped onto the test paper of the invention, and the surface-enhanced Raman spectrum of the test paper is detected by a Raman spectroscopy analyzer, and finally the Raman spectrum is enhanced by the surface. The spectrum can be interpreted as the type of the molecule to be tested.

In the past, the substrate used for Raman spectroscopy was a hard substrate, and the drop was contained. After the solution of the molecule is measured, since the substrate does not absorb water, the solution forms water droplets on the substrate, and the curved surface of the water droplet due to surface tension affects the measurement of the Raman spectrum.

Since the test paper of the present invention is a water absorbing material, the solution is absorbed by the test paper after the test paper is dropped thereon, and the drop-like shape is not formed on the surface to affect the analysis of the Raman spectrum. In addition, referring to FIG. 2, since the test paper has the 3D structure 202 formed by the micro or sub-micrometer fiber structure 201, the molecule to be tested (for example, the biological macromolecule 204 or the compound 205) in the solution 203 can be adsorbed, The solution molecules (for example, water molecules) are allowed to flow away from the gaps of the fiber structure 201, so that the fiber structure also has the effect of concentrating the molecules to be tested. The test paper of the present invention is capable of detecting a more sensitive range, for example, a concentration of 100 nM can be detected using a 1 μl volume of the test solution.

In addition, the Raman test paper can be surface-modified as required. For example, after step 104 or step 105, the surface of the substrate is modified to increase the accuracy of the molecule to be tested; or the antibody is coated on the surface to detect the biological molecule to be tested. For example, detecting antigens, antibodies or viral particles.

The method for manufacturing the portable micro-Raman optical test strip of the invention has the characteristics of convenient production and ability to achieve micro analysis. Furthermore, since no other equipment is required in the manufacturing process, the size and shape of the Raman optical inspection test paper produced by applying the manufacturing method of the present invention are not limited by the apparatus. Therefore, it can be applied to water quality inspection, food safety, microbial detection, cancer cell calibration technology, and the like.

[Example 1]

Filter paper (ADVANTIC, thickness 200μm) was cut into 6mm diameter with a puncher The circle was mixed in an equal volume with 10 mM TNA (tannic acid) as a reducing agent and experimental ethanol, and the filter paper was immersed in a reducing agent. The excess reducing agent was then removed and dried using a 50 ° C drying vessel for 5 minutes. 6 μl of a 5 mM chloroauric acid solution was added dropwise, and dried using a drying vessel at 50 ° C for 5 minutes. The obtained test paper was analyzed by a scanning electron microscope (model). Figure 3A shows an image of the front side of the test paper (ie, the chloroauric acid drop-in surface), and it can be observed that nano- or sub-micron fibers are formed with nano metal particles (white spots in the image), and the particle size distribution is about 80-120 nm; Fig. 3B shows an image of the back side of the test paper (i.e., the other side of the chloroauric acid dropping surface), and it was also observed that the nano gold particles were formed on the fiber. From the above results, it was found that the Raman test paper having the nano gold particles on the surface of the test paper to the inside or even the back surface can be produced by using the test paper production method of the present invention.

[Embodiment 2]

The filter paper (ADVANTIC) was cut into a 6 mm diameter circle by a puncher, five circular filter papers were stacked, 10 mM TNA as a reducing agent and experimental ethanol were mixed in equal volume, and the stacked filter paper was immersed in a reducing agent. in. The excess reducing agent was then removed and dried using a 50 ° C drying vessel for 5 minutes. 6 μl of a 5 mM chloroauric acid solution having a pH of 2.35 was dropped, and dried using a drying vessel at 50 ° C for 5 minutes.

[Comparative Example 1]

The same procedure as in Example 2 was carried out except that the immersion reducing agent in Example 2 was changed to spray the reducing agent from above to the filter paper.

The products obtained in Comparative Example 1 and Example 2 were separated from the top to the bottom to observe whether or not dark nano-gold particles were formed on the surface of the filter paper. As shown in the results of Fig. 4, in Comparative Example 1 in which a reducing agent was sprayed, only the test papers of the first layer and the second layer had grown nanoparticles of nanoparticles. In contrast, the use of soaking reducing agents In Example 2, nano gold particles were grown in the first layer to the fifth layer. From the above results, it is understood that the test paper of the present invention can grow nano-gold particles on the surface to the inside or even the back surface of the test paper.

[Example 3]

The filter paper (ADVANTIC) was cut into a 6 mm diameter circular shape by a puncher, 10 mM TNA as a reducing agent and experimental ethanol were mixed in an equal volume, and the filter paper was immersed in a reducing agent. The excess reducing agent was then removed and dried using a 50 ° C drying vessel for 5 minutes. 6 μl of a 5 mM chloroauric acid solution having a pH of 2.35 was dropped, and dried using a drying vessel at 50 ° C for 5 minutes.

[Comparative Example 2]

The same procedure as in Example 3 was carried out except that the immersion reducing agent in Example 3 was changed to spray the reducing agent from above to the filter paper.

Whether or not the test paper obtained in Comparative Example 2 and Example 3 was formed with nano gold particles was observed. As shown in the results of Fig. 5, the fifth (A) is a comparative example 2 in which a reducing agent was sprayed, and the nano gold particles grown on the surface of the filter paper showed an uneven distribution. In contrast, Figure 5(B) shows Example 3 using a soaking reducing agent with a uniform distribution of nanogold on the surface of the test paper.

[Example 4]

The filter paper (ADVANTIC) was cut into a 6 mm diameter circular shape by a puncher, 10 mM TNA as a reducing agent and experimental ethanol were mixed in an equal volume, and the filter paper was immersed in a reducing agent. The excess reducing agent was then removed and dried using a 50 ° C drying vessel for 5 minutes. Thereafter, 6 μl of a chloroauric acid solution having a pH of 2.35 and a concentration of 1, 3, and 5 mM, respectively, was dropped, and dried using a drying vessel at 50 ° C for 5 minutes.

Observe the test paper obtained, when the concentration of chloroauric acid is 1~5mM, on the test paper The fiber structure produces nano gold particles.

[Example 5]

The filter paper (ADVANTIC) was cut into a 6 mm diameter circle by a puncher. A TNA solution having a concentration of 0.1, 1, 10, 20, 30, 40, 50 mM as a reducing agent was prepared, and TNA and experimental ethanol were mixed in an equal volume, and the filter paper was immersed in a reducing agent. The excess reducing agent was then removed and dried using a 50 ° C drying vessel for 5 minutes. Thereafter, 6 μl of a chloroauric acid solution having a pH of 2.35 and a concentration of 5 mM was dropped, followed by drying the test paper at 50 °C.

When the test paper obtained was observed, when the TNA concentration was more than 0.1 mM, nano gold particles were formed in the fiber structure of the test paper. Among them, when the TNA concentration is 1~20mM, the nano-gold particles can be formed in the test paper fiber structure; while the TNA concentration is 30~50mM, although the nano-particles of the test paper fiber structure are few, the nano-gold can still be formed. Particles.

Then, 0.1 mM methylene blue solvent as a molecule to be tested was prepared, and Raman spectroscopy analysis was used to detect the Raman spectrum of methylene blue, and it has the function as a Raman optical test strip.

[Embodiment 6]

The filter paper (ADVANTIC) was cut into a 6 mm diameter circle by a puncher. A tannic acid (TNA) solution as a reducing agent was prepared at a concentration of 10 mM, and the filter paper was immersed in a reducing agent. The excess reducing agent was then removed and dried using a 50 ° C drying vessel for 5 minutes. Thereafter, 6 μl of a chloroauric acid solution having a pH of 2.35 and a concentration of 5 mM was added thereto, and the drying vessel temperature was set to 4 ° C, 25 ° C, 50 ° C, 75 ° C, and 100 ° C, respectively, and dried for 5 minutes.

Observe the test paper obtained, when the drying temperature is 4~100 °C, on the surface of the test paper Both produce nano gold particles.

Then, 0.1 mM methylene blue solvent as a molecule to be tested is prepared, and the Raman test paper prepared by Raman spectroscopy analysis and drying at 4 to 100 ° C can detect the Raman spectrum of methylene blue. The function of Raman optical test strips.

[Embodiment 7]

The filter paper (ADVANTIC) was cut into a 6 mm diameter circle by a puncher. A tannic acid (TNA) solution as a reducing agent was prepared at a concentration of 10 mM, and the filter paper was immersed in a reducing agent. The excess reducing agent was then removed and dried using a 50 ° C drying vessel for 5 minutes. The pH of the 5 mM chloroauric acid solution was adjusted to 2.35, 3.21, 4.1, 5.14, respectively, using NaOH. Thereafter, 6 μl of a 5 mM chloroauric acid solution having a different pH was dropped, and the drying vessel temperature was set to 50 ° C for 5 minutes.

Then, a 0.1 mM methylene blue solvent as a molecule to be tested is disposed, and Raman ray paper prepared by a Raman spectroscopy analyzer having a pH of 2 to 7 of a chloroauric acid solution can detect a Raman spectrum of methylene blue. It has the function as a Raman optical test strip.

[Embodiment 8]

The filter paper (ADVANTIC) was cut into a 6 mm diameter circle by a puncher. A tannic acid (TNA) solution as a reducing agent was prepared at a concentration of 10 mM, and the filter paper was immersed in a reducing agent. The excess reducing agent was then removed and dried using a 50 ° C drying vessel for 5 minutes. Thereafter, 6 μl of a chloroauric acid solution having a pH of 2.35 and a concentration of 5 mM was dropped, and the drying vessel temperature was set to 50 ° C for 5 minutes.

[Comparative Example 3]

Using a commercially available Raman substrate, for example, a Raman substrate manufactured by Hekang Biotech, a nanoporous hole (5 nm pitch) of alumina is used as a substrate on a glass hard substrate, and 25 nm of nano silver particles are placed in the hole. Its Raman signal amplification efficiency can reach 10 ¹¹ . After opening, in order to avoid substrate oxidation, it needs to be used within 3-5 days.

0.01 mM 4-aminothiophenol and 0.1 mM MG were separately disposed as the molecules to be tested, and 1 μl of 4-aminothiophenol or 1 μl of MG was dropped onto the Raman test papers of Example 8 and Comparative Example 3, respectively. The obtained Raman spectrum is shown in Fig. 6 by analysis by a Raman spectrum analyzer. From the results of FIGS. 6(A) and 6(B), it can be seen that a Raman test paper using a commercially available Raman substrate, as shown in FIG. 6(A), cannot detect 0.01 mM of 4-amine at 785 nm. Raman spectroscopy of thiophenol, in contrast, using the Raman test paper of the present invention, as shown in Fig. 6(B), the Raman spectrum of 0.01 mM 4-aminothiophenol can be detected at 785 nm. It has the function as a Raman optical test strip.

Further, from the results of the sixth (C) and the sixth (D), it is understood that the Raman test paper (Fig. 6C) using a commercially available Raman substrate cannot detect the Raman of 0.1 mM MG at 785 nm. In the spectrum, in contrast, the Raman test paper of the present invention, as shown in Fig. 6(D), can detect a Raman spectrum of 0.1 mM MG at 785 nm, and has a function as a Raman optical test strip.

[Embodiment 9]

The Raman test paper of the present invention was produced in the same manner as in Example 8. Then, a methylene blue solution having a concentration of 100 nM, 1 μM, and 10 μM, respectively, was set as a molecule to be tested, and 1 μl of the methylene blue solution was dropped onto the Raman test paper of Example 9, and then analyzed by a Raman spectrum analyzer to obtain the obtained Raman spectrum. display In Figure 7. The Raman test paper of the present invention is capable of detecting a Raman spectrum of a methylene blue solution having a concentration of 100 nM. That is, the sensitivity of the Raman test paper of the present invention is a concentration range in which the nM level can be detected in a volume of μl.

From the above results, it is understood that the Raman optical test strip produced by the method of the present invention has the characteristics of simple manufacture and high detection sensitivity, and can detect a concentration range of nM level in a volume of μl.

Claims (8)

A method for manufacturing a portable Raman optical test strip comprising the steps of: (a) providing a substrate, wherein the substrate is a water absorbent material having a micron or submicron fiber structure; and (b) soaking the substrate to a reducing agent for attaching the reducing agent to the micro or sub-micron fiber structure; and (c) dropping a metal salt solution to the substrate to react the metal salt solution with the reducing agent, and at the micron or Nano metal particles are grown on the submicron fiber structure. The method for manufacturing a portable Raman optical test strip according to claim 1, further comprising repeating the steps (b) and (c) more than once. The method for manufacturing a portable Raman optical test strip according to the first aspect of the patent application, after the step (b) and/or the step (c), further comprises a drying step, wherein the drying step is 40~ It lasts for 1 to 10 minutes at 80 °C. The method for producing a portable Raman optical test strip according to claim 1, wherein the water absorbing material comprises paper, sponge, cotton, or a combination thereof. The method for producing a portable Raman optical test strip according to claim 1, wherein the reducing agent is a single type of natural reducing agent, and the reducing agent has a concentration ranging from 1 to 50 mM. The method for manufacturing a portable Raman optical test strip according to claim 1, wherein the reducing agent is natural tea polyphenol, curcumin, Citric acid, or vitamin C. The method for manufacturing a portable Raman optical test strip according to claim 1, wherein the metal salt solution is a chloroauric acid solution, a silver nitrate solution or a combination thereof, wherein the chloroauric acid solution has a concentration ranging from 1 to 10 mM. The silver nitrate solution has a concentration ranging from 0.001 to 15 mM. The method for manufacturing a portable Raman optical test strip according to claim 1, further comprising subjecting the substrate to functional group modification or surface coating of the substrate to perform surface modification after the step (c).