TWI496194B - Flexible surface enhanced raman substrate - Google Patents

Description

Flexible surface enhanced Raman spectroscopy substrate

The invention relates to a substrate which can be used for detecting surface enhanced Raman spectroscopy of microorganisms, in particular to a flexible Raman substrate of silver nanoparticle/nano clay enamel.

Surface-enhanced Raman spectroscopy (SERS) is mainly to obtain Raman spectra of samples adsorbed on the surface of colloidal metal particles (usually silver, gold or copper) or on the rough surface of such metal sheets to obtain higher Raman spectra. Raman spectral signal. Surface Raman spectroscopy (SERS) has been extensively studied in biological systems due to the small sample size required for Raman spectroscopy, the extremely low sensitivity to water interference, and the sensitivity to configuration and the environment. application.

Since the surface-enhanced Raman substrate (SERS substrate) exhibits 10 ⁶ to 10 ¹³ times Raman signal enhancement and can be detected without label-free, it can be used as a microorganisms such as bacteria. For testing purposes. Inventor Professor Wang Yulin has successfully used this high-stability surface-enhanced Raman substrate (Ag/AAO array glass substrate) for a series of detection and drug resistance tests on bacteria. In addition, the inventors also tried to immobilize the glycopeptide on the surface-enhanced Raman substrate, making the substrate selective for bacteria in clinical samples, and having the functions of capturing bacteria and detecting Raman spectroscopic signals. However, if this technology is to be widely used in clinical trials and living environment, for some irregular and large surfaces, such as fungi, molds and cancer cells, it is limited by microbes and surface-enhanced Raman substrate stickers. Attached to the problem and the space limitation of the surface-enhanced Raman amplification effect.

SUMMARY OF THE INVENTION A primary object of the present invention is to provide a substrate for flexible surface enhanced Raman spectroscopy which can improve the space limitation of conventional conventional substrates for microorganisms and increase the contact area with microorganisms.

A second object of the present invention is to provide a flexible surface-enhanced Raman spectroscopy substrate which can effectively detect microorganisms having irregular surfaces, larger microorganisms, and microorganisms having hydrophilicity.

A third object of the present invention is to provide a substrate for flexible surface enhanced Raman spectroscopy which can effectively enhance Raman signals.

The substrate for flexible surface enhanced Raman spectroscopy provided by the present invention comprises a delaminated citrate clay carrier and a plurality of metals fixed to the delaminated citrate clay carrier. particle.

The delaminated citrate clay may be selected from at least one of bentonite, lithium bentonite, montmorillonite, synthetic mica, kaolin, talc, attapulgite, vermiculite or layered double hydroxide. The delaminated flaky clay is preferably selected from the montmorillonite delamination.

The metal particles described above may be selected from one of materials such as silver particles, copper particles, iron particles or gold particles, and preferred materials are silver particles.

In order to achieve the above object, another flexible surface enhanced Raman spectroscopy substrate provided by the present invention further comprises a surfactant.

The above surfactant nonionic surfactant is preferably SINOPOL 1830 (polyoxyethylene stearylcetyl ether).

To achieve the above object, the present invention provides a method for manufacturing a flexible surface enhanced Raman spectroscopy substrate by mixing a delaminated citrate clay carrier solution with a metal ion solution to carry out a reduction reaction. The metal ions are reduced to metal particles and attached to the delaminated tantalate clay carriers, respectively.

Wherein, before the delaminated citrate clay carrier solution is mixed with the metal ion solution, the delaminated citrate clay carrier solution is mixed with a surfactant solution to perform a mismatch reaction, thereby obtaining the Surfactant modified delaminated citrate clay carrier.

Thereby, various types of pro-hydrophobic microorganisms can be detected by using the substrate of the flexible surface-enhanced Raman spectrum. Moreover, the flexible surface-enhanced Raman spectroscopy substrate has flexibility, and can improve the space limitation and poor adhesion of the conventional substrate for microbial detection. In addition, since the thickness of the nano-clay sheet is only about one nanometer, the silver nano-particles can be adsorbed on both sides, which can cause very large Raman amplification hot spots in the z direction compared with the conventional substrate. With the xy direction hotspot, it can form a Raman magnifying hotspot in three dimensions, and its Raman magnification is considerable.

The following is an experiment conducted by the present invention to explain the raw materials and strain sources used in the respective examples, including:

1. Nanoscale Silicate Platelets (NSP): can be obtained by delamination of sodium ion montmorillonite (Na ⁺ -MMT). For the preparation method, please refer to the Republic of China Announcement Nos. I280261, I284138, I270529, 577904 and 593480 equal invention patent.

2. Silver nitrate _{(AgNO 3):. Mw =} 169.87g / mol, available from JTBaker, Inc ..

3. Ethanol (EtOH): 99.5%, is a weak reducing agent. When it is 30~150°C, silver ions can be slowly reduced to nano silver.

4. Diethanolamine (DEA): HN(CH ₂ CH.OH) ₂ , a weak reducing agent that slowly reduces silver ions to nano silver.

5. SINOPOL 1830: Polyoxyethylene stearylcetyl ether, a polyoxyethylene alkyl ether purchased from Sino-Japanese Synthetic Chemical Company.

6. Species: Staphylococcus aureus 71 (431; 1078) and Escherichia coli are wild isolates provided by Professor Su Honglin, Department of Life Sciences, Zhongxing University.

The above is a description of the materials provided in the preferred embodiments of the present invention, and many examples are described to illustrate the embodiments of the present invention, but these examples are for illustrative purposes only, and those skilled in the art will understand that these examples are not exhaustive and not expected. It is intended to limit the scope of the invention.

A preferred embodiment of the flexible Raman-enhanced (SERS) substrate of the present invention is The composition of the nano silver particles/nano clay slabs is described, but the flaky clay obtained by delamination of different clays may be used as a carrier, and the clays may be bentonite, lithium bentonite, montmorillonite, Synthetic mica, kaolin, talc, attapulgite, vermiculite or layered double hydroxide.

The metal ions of the embodiments of the present invention are not limited to silver ions and may be gold, copper, iron or other suitable metals. Further, the silver ion source is not limited to silver nitrate as long as it can appropriately supply silver ions, for example, a silver bromide solution, a silver chloride solution, a silver bromate solution or a silver chlorate solution.

The surfactant in the embodiment of the present invention is not limited to SINOPOL 1830, and may also be the following nonionic surfactants, such as polyoxyrthylene alkyl ether, sorbitol fatty acid (span), and polysorbate (polysorbate). Tween), alkylphenol ethoxylates, nonylphenol ethoxylates (NPEOs), fatty alcohol ehtoxylates.

A preferred synthetic embodiment of a flexible Raman-enhanced (SERS) substrate of the present invention comprises two parts:

(1) Synthetic steps of silver (Ag/NSP).

(2) Synthetic step of surfactant silver (Ag/NSS).

Example 1. Synthesis step of silver (Ag/NSP)

Prepare a delaminated clay bract (NSP) solution (46.5 g, 2 wt% in water) and a silver nitrate (AgNO ₃ ) solution (0.11 g, 1 wt%), then place 2 wt% of the delaminated clay bract (NSP) solution In a 250 mL three-necked round bottom flask, an ethanol solution (49.5 g) was added to make the final concentration of the solution 1 wt%, and the magnet was stirred for half an hour, and nitrogen gas was introduced into the reaction apparatus to maintain the system under nitrogen atmosphere to avoid oxidation. Silver is generated; in addition, a condensing reflux device is installed to avoid evaporation of ethanol at high temperature, and then a 1 wt% silver nitrate (AgNO ₃ ) solution is slowly dropped into the solution, and stirring is continued for half an hour, at which time the solution is beige and finally heated to 80. °C, the solution will start the redox reaction, the color will change slowly. When the reaction is 3 hours, the solution will appear yellowish brown. During the reaction, the formation of silver nanoparticles will be monitored by UV/Vis spectrometer. The characteristic absorption wavelength of the silver-silver particles is 408 nm), and the reaction-intensity value does not change. The reaction ends. At this time, the excess unreacted ethanol is filtered by suction filtration (using whatman® No. 5 filter paper, Cat. No. 1005 090). Off, will be produced again Scraped from the filter back to the storage solution (3wt%) in water, is the product of this Xipian silver (Ag / NSP), when the Xipian silver (Ag / NSP) was diluted to 100 ppm, a golden yellow solution.

Example 2. Synthetic step of surfactant silver (Ag/NSS)

The delaminated clay flakes (NSP) (25 g, 1 wt% in water) are mixed with the surfactant SINOPOL 1830 (25 g, 1 wt% in water) and the like, and the magnet is stirred at room temperature for half an hour to carry out a mismatch reaction. The product of the delaminated clay flakes (NSP) modified with the surfactant SINOPOL 1830 was obtained, which was named here as NSS.

Next, the NSS solution (50 g, 1 wt% in water) was placed in a 100 mL three-necked round bottom flask and stirred with a magnet for half an hour, and nitrogen was passed through to maintain the system in a nitrogen atmosphere to avoid the formation of silver oxide; Silver nitrate (AgNO ₃ ) solution (5.9 g, 1 wt% in water) was slowly dropped into the NSS solution, stirring was continued for 10 minutes, then diethanolamine solution (0.8 g, 10 wt% in water) was added, stirring was continued for 10 minutes, and finally the temperature was raised to At 50 ° C, during the reaction, the formation of nano silver particles was monitored with an ultraviolet/visible spectrometer over time, and the reaction intensity value was no longer changed, and the reaction was terminated, and the surfactant silver (Ag/NSS) was obtained.

The above is a material configuration and synthesis method of a preferred synthesis embodiment of the present invention, and then the system apparatus for measuring Raman spectroscopy of the present invention and the measurement sample configuration are explained.

Raman system equipment

Raman spectroscopy measurements were performed using a Model HR800, Horiba Raman microscope to observe the scattering from the 632.8 nm 氦氖 laser. The laser light is focused on the sample with a 50x objective. Spectral analysis was performed using the same objective lens to collect the reverse scattered light. The Raman spectrum detection time is 60 seconds, and the detection frequency ranges from 400 to 1800 cm ^-1 . (With a 100x water mirror on the device, the SERS spectrum of a high quality single bacteria will be obtained in 1-3 seconds.)

Measuring sample configuration

A. The Ag/NSP solution and the Ag/NSS solution are respectively set to 100 ppm/ml; B. The bacterial solution to be tested is configured to be 10 ⁸ cells/ml; C. The Ag/NSP solution and the test solution to be tested are in a volume of 1: Mixing ratio of 1 (vol%); mixing Ag/NNS solution and the solution of bacteria to be tested in a ratio of 1:1 (vol%); D. mixing the solution after mixing in step C for a short period of time, mixing and mixing The droplets are placed on an aluminum sheet and, after drying, can be placed under Raman spectroscopy for testing.

Next, the implementation and efficacy of the flexible surface-enhanced Raman substrate of the present invention for detecting microbial molecular fingerprints are further explored. The preferred embodiment includes three parts:

(1) The ratio of silver nanoparticle (Ag) to tantalum (NSP) causes the influence of Ag/NSP SERS wafers.

(b) Differences between Ag/NSP and Ag/NSS substrates for hydrophilic and hydrophobic bacteria.

(3) Application of Ag/NSS substrates to larger microorganisms (fungi) and superhydrotomycobacteria.

[The effect of silver nanoparticle (Ag) and nano-slice (NSP) ratio on Ag/NSP SERS wafers]

The invention prepares six groups of different weight ratios of silver nanoparticles/nano-nano sheets (Ag0/NSP100, Ag1/NSP99, Ag7/NSP93, Ag15/NSP85, Ag30/NSP70, Ag50/NSP50) to fabricate a flexible SERS substrate. The above six ratios were tested with Staphylococcus aureus to test the optimal SERS detection conditions (including enhancement magnification and stability). As shown in Figure 1, the Ag15/NSP85, Ag30/NSP70, and Ag50/NSP50 groups can all achieve good SERS signals (integral 730cm ^-1 peak area to infer the signal), although the Ag50/NSP50 group sometimes A very strong signal appears, but the stability is best for the AgS/NSP70 SERS substrate, and the subsequent experiment will take an Ag/NSP ratio of 30/70 (wt%).

[The difference between Ag/NSP and Ag/NSS substrates for hydrophilic and hydrophobic bacteria]

Next, the difference between the Ag/NSP and Ag/NSS substrates of the present invention for detecting hydrophilic and hydrophobic bacteria is discussed. Please refer to the lines (a) and (b) in Figure 2, (a) for the Ag/NSP substrate and the hydrophilic S. aureus (SA) test results, the Ag/NSP substrate can be obtained for the more hydrophilic bacteria. Good SERS signal; (b) Test results of Ag/NSP substrate and hydrophobic Escherichia coli (EC), the Ag/NSP substrate is not sensitive to SERS detection of more hydrophobic bacteria. Next, please refer to the line (d) of Fig. 2, which is the test result of Ag/NSS substrate and Escherichia coli (EC). It is found that the SERS signal of the hydrophobic bacteria can be improved by adding the Ag/NSS substrate of the surfactant. Comparing Figures 2(d) and (b), it is apparent that the SERS signal of hydrophobic E. coli increases the number of SERS signals by several tens of times after the addition of the surfactant.

[Application of Ag/NSS substrate for larger microorganisms (fungi) and superhydrotoxin]

Figure 3 is a spectrum diagram of a larger microbial-fungus and superhydrophobic bacteria-mycobacteria reacted with a surfactant silver-Ag/NSS substrate. Confirming the Ag/NSS substrate with surfactant, A stable and sensitive SERS spectrum can be obtained. The flexible SERS substrate of the present invention is expected to be useful for detecting various microorganisms (as small as viruses, bacteria, as large as fungi, cancer cells, etc.).

In summary, referring to FIG. 4, the flexible SERS substrate 1 of the preferred embodiment of the present invention replaces the nano silver particle (AgNP) 12 with a nano-silicate platelet by ion exchange. The NSP)11 surface is formed to form an Ag/NSP flexible SERS substrate 1, and the Ag/NSP flexible SERS substrate 1 is adsorbed on the surface of a sample 2 by the chargeability of the surface of the delaminated clay sheet 11. Then, the laser light S1 is used to focus on the sample 2, and the excited scattered light S2 is collected and subjected to SERS spectral analysis. The sample 2 may be a microorganism or a cell such as a bacterium, a fungus or the like.

The above flexible SERS substrate has the following advantages over the general SERS substrate: (1) it is easy to attach to irregularly shaped microorganisms or cells by the flexibility of the delaminated clay plaque (about one nanometer in thickness); (2) Enhancing the characteristics of the Raman signal by the nano silver particles; (3) Having the features of the Raman enhancement hot spots in the three-dimensional direction. Generally, SERS substrates have only Raman hotspots in the x-y direction. Referring to FIG. 5, a flexible SERS substrate 1 according to a preferred embodiment of the present invention includes a delaminated clay crucible 11 and silver nanoparticles 12. Wherein, the delaminated clay crucible 11 has double-sided adsorbability, so that the double-sided can simultaneously adsorb the silver nanoparticle 12, and the distance D1 of the adjacent silver nanoparticle 12 in the x-y direction is between 1 and 20 The spacing D2 of the adjacent silver nanoparticles 12 in the z direction is between 1 and 2 nm. As shown in the transmission electron micrograph of Fig. 6, it can be clearly verified that the silver nanoparticle 12 is adsorbed on both sides of the delaminated clay crucible 11. Further, the thickness of the delaminated clay sheet 11 is only about 1 nm (having flexibility and optical transparency), and therefore, as shown in Fig. 5, the hot spot H2 in the z direction becomes quite large (when the particle distance is closer) When not touching, the hotspot is stronger, and then the hot spot H1 in the xy direction can form a Raman magnifying hotspot in three dimensions, resulting in a considerable Raman magnification, which is much better than the traditional Raman signal.

By virtue of the above advantages, the flexible SERS substrate 1 can easily obtain the "molecular fingerprint" of these microorganisms or cells for the purpose of rapid screening.

In addition, the present invention also performs some polymer-polymerized hydrophilic-hydrophobic modification on the surface of the silver (Ag/NSP) or a surfactant-forming agent (Ag/NSS) by adding a polyoxyethylene alkyl ether. It is widely used in the detection of various types of hydrophilic and hydrophobic microorganisms and cells.

The above description is only for the preferred embodiments of the present invention, and the equivalent structures and manufacturing methods of the present invention and the scope of the patent application are intended to be included in the scope of the present invention.

1‧‧‧Flexible SERS substrate

11‧‧‧Delamed clay sheet

12‧‧‧ Silver Nanoparticles

2‧‧‧ samples

S1‧‧‧Laser light

S2‧‧‧scattered light

D1‧‧‧x-y direction adjacent silver nanoparticle spacing

Adjacent silver nanoparticle spacing in the D2‧‧‧z direction

Hotspots in the H1‧‧x-y direction

Hotspots in the direction of H2‧‧‧z

Figure 1 is a Raman spectrum showing the effect of the weight ratio of silver nanoparticles (Ag) and tantalum (NSP) on the Ag/NSP SERS wafer (the number represents wt%, and the spectrum of each group is 5 samples). Figure 2 is a Raman spectrum showing the effect of an Ag/NSS substrate with surfactant added on hydrophilic bacteria (SA, Staphylococcus aureus) and hydrophobic bacteria (EC, E. coli); Figure 3 is Raman spectroscopy , showing the SERS spectrum of the Ag/NSS substrate with surfactant added to fungus (Fungus) and Mycobacterium; FIG. 4 is a schematic diagram of attaching the sample to the flexible SERS substrate; FIG. 5 is a three-dimensional Raman of the flexible SERS substrate. A schematic diagram of the enlarged hot spot; Figure 6 is a transmission electron micrograph showing that the silver nanoparticles are adsorbed on both sides of the nano-clay sheet.

1‧‧‧Flexible SERS substrate

11‧‧‧Delamed clay sheet

12‧‧‧ Silver Nanoparticles

2‧‧‧ samples

S1‧‧‧Laser light

S2‧‧‧scattered light

Claims (18)

A flexible surface enhanced Raman spectroscopy substrate comprising a delaminated citrate clay support, and a plurality of metal particles affixed to the delaminated citrate clay support, wherein the delaminated tellurite clay The carrier is a flaky clay selected from the group consisting of bentonite, lithium bentonite, montmorillonite, synthetic mica, kaolin, talc, attapulgite, vermiculite and layered double hydroxide. The delaminated flaky clay is a nano delamination delaminated from montmorillonite, the aspect ratio of which is 100-1000, and has flexibility; the characteristics of the substrate of the flexible surface enhanced Raman spectroscopy In order to have a three-dimensional Raman amplification hot spot to enhance the Raman effect. The substrate of the flexible surface-enhanced Raman spectrum according to claim 1, wherein the metal particles are at least one selected from the group consisting of silver particles, iron particles, copper particles, and gold particles. The substrate of the flexible surface enhanced Raman spectrum of claim 1, wherein the metal particles are silver particles. The substrate of the flexible surface-enhanced Raman spectrum according to claim 1, which is characterized in that it is used as a microorganism for detecting irregular surfaces or detecting a larger type of microorganism. A flexible surface enhanced Raman spectroscopy substrate comprising a delaminated citrate clay support, a plurality of metal particles affixed to the delaminated citrate clay support, and a plurality of ruthenium fixed to the delamination a surfactant of a clay carrier, wherein the delaminated citrate clay carrier is selected from the group consisting of bentonite, lithium bentonite, montmorillonite, synthetic mica, kaolin, talc, attapulgite, vermiculite and layered At least one of the double hydroxides is delaminated flaky clay, wherein The delaminated flaky clay is a nano-striped smear from montmorillonite with an aspect ratio of 100-1000 and flexibility; the flexible surface-enhanced Raman spectroscopy substrate is characterized by Raman magnifies hot spots in three dimensions to enhance the Raman effect. The substrate of the flexible surface-enhanced Raman spectrum according to claim 5, wherein the metal particles are at least one selected from the group consisting of silver particles, iron particles, copper particles, and gold particles. The substrate of the flexible surface enhanced Raman spectrum of claim 5, wherein the metal particles are silver particles. The substrate of the flexible surface enhanced Raman spectrum of claim 5, wherein the surfactant is a nonionic surfactant. The substrate of the flexible surface enhanced Raman spectrum of claim 5, wherein the surfactant is SINOPOL 1830 (polyoxyethylene stearylcetyl ether). A substrate for flexible surface enhanced Raman spectroscopy according to claim 5, which is characterized by being used as a microorganism for detecting hydrophobic properties. A method for producing a substrate for flexible surface enhanced Raman spectroscopy according to claim 1, wherein a delaminated citrate clay carrier solution is mixed with a metal ion solution to carry out a reduction reaction to reduce the metal ion Forming metal particles and respectively adhering to the delaminated citrate clay carrier, wherein the delaminated citrate clay carrier is selected from the group consisting of bentonite, lithium bentonite, montmorillonite, synthetic mica, kaolin, talc a flaky clay obtained by delaminating at least one of attapulgite, vermiculite and layered double hydroxide, wherein the delaminated flaky clay is a glutinous rice delaminated from montmorillonite. The aspect ratio is 100~1000, and Flexible; the flexible surface enhanced Raman spectroscopy substrate is characterized by three-dimensionally directed Raman amplification hot spots. The method for producing a substrate of a flexible surface enhanced Raman spectrum according to claim 11, wherein the delaminated tannic acid is first mixed before the delaminated tellurite clay carrier solution is mixed with the metal ion solution. The salt clay carrier solution is mixed with a surfactant solution to carry out a mismatch reaction to obtain a delaminated citrate clay carrier having the surfactant modification. The method for producing a substrate of a flexible surface-enhanced Raman spectrum according to claim 11, wherein the metal ion is at least one selected from the group consisting of silver ions, iron ions, copper ions, and gold ions. The method for producing a substrate of a flexible surface-enhanced Raman spectrum according to claim 11, wherein the metal ion is silver ion. The method for producing a substrate of a flexible surface enhanced Raman spectrum according to claim 11, wherein the metal ion solution is selected from the nitrate solution of the metal ion, the chloride solution and the bromide solution. At least one. The method for producing a nanocomposite capable of controlling the volume of a metal particle according to claim 15, wherein the metal ion is silver ion, and the metal ion solution is selected from the group consisting of a silver nitrate solution, a silver chloride solution and a silver bromide solution. At least one of them. The method for producing a nanocomposite capable of controlling the volume of a metal particle according to claim 12, wherein the surfactant is a nonionic surfactant. The method for producing a nanocomposite capable of controlling the volume of a metal particle according to claim 12, wherein the surfactant is SINOPOL 1830 (polymerized) Ethylene glycol octadecyl ether, polyoxyethylene stearylcetyl ether).