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Abstract
Irregular substrates are inappropriate for enhancing surface enhanced Raman scattering (SERS) due to their poor performances in terms of uniformity, enhancement performance, and polarization characteristics. However, in this work, we purposely employed a natural biological razor clam material with messy and irregular structures to improve the SERS. The rough surface was achieved by magnetron sputtering Ag nanoislands on the prism layer of the razor clams, and the Ag nanoparticles were treated using the method of oil-water interface self-assembly to form relatively uniform structures. Compared to the substrate without Ag nanoparticles, the presented substrate has better reproducibility, polarization-independence, and higher SERS intensity, and the detect limitation of R6G can be decreased from 10−12 M to 10−18 M. The ultrasensitive detection of thiram gives our structures potential for high sensitivity biosensors.
© 2021 Optical Society of America under the terms of the OSA Open Access Publishing Agreement
1. Introduction
Surface-enhanced raman scattering (SERS) is a powerful, effective, and convenient analytical tool widely used for rapid, sensitive, and non-destructive detecting various analyte molecules [1–3]. It has been widely used in medical biology [4,5], environmental monitoring [6], food-safety testing [7,8], and trace detection [9,10]. SERS refers to the phenomenon that the Raman scattering signal can be greatly enhanced when molecules are distributed on the nanoscale rough metal surface. Generally, electromagnetic enhancement (EM) and chemical enhancement (CM) were employed as two basic methods of enhancing SERS. In case of EM, the localized surface plasmon resonance (LSPR) the collectively oscillating resonant electrons, have been considered as an important platform to improve SERs. For CM, charge transfer (CT) is the main reason of enhanced SERS. In many cases, a combination of these two mechanisms contribute to the SERS enhancement. Theoretical and experimental results have confirmed that EM plays a major role in enhancing the intensity of Raman signal at 104-1012, while CM only improve the Raman signal around 101-102 [11]. Our research group has been focused on exploring the substrate's morphology, structures or material variations on enhancing SERS. In order to illustrate the local field enhancement characteristics, we employed 3D-FDTD to simulate the substrate, and also marked the location of the possible “hot spots”. Therefore, in this work, EM is more important. SERS substrates usually choose noble metals such as gold (Au), silver (Ag), platinum (Pt), because their LSPR can be induced in a broad wavelength range covering visible light and near-infrared [12–14]. Compared to other metal materials, Ag has relatively better stability and higher sensitivity, and it is widely used in the preparation of SERS substrates [15]. Therefore, we choose Ag as the metal material for the preparation of SERS substrate with high performance and low detection limit.
In general, substrates with regular structures are preferred, and those with irregular structures are mostly ignored. Because regular structural substrates, such as nanopillars [16,17], graphene [18–21] and carbon nanotubes [22], can effectively increase the density of “hot spots” and detect molecular binding sites [23], leading to enhanced Raman signal, polarization, sensitivity and other optical properties. However, instead of employing traditional selection for relatively regular substrate, here we choose an irregular substrate and make it more regular in order to improve the corresponding SERS performances. The oil-water separation self-assembly method is employed to modify the Ag nanoparticles on the substrate by following procedures: using ethanol as an inducer, self-assembling Ag nanoparticles at the interface between N-Hexane and water, and forming a two-dimensional liquid metallic Ag mirrorlike nano-film. Then, we use the pulling method to modify the substrates with Ag nanoparticles. The amplified SERS signal of most noble metals substrates has a good relationship with the relative orientation between the incident polarization and the enhancement direction [24]. In this way, we can judge whether the substrate is uniform based on the polarization dependence or independence of the substrate. Magnetron sputtering was used to modify the Ag nanoislands onto the substrates, and the Ag nanoparticles layer is modified onto the irregular substrates with the method of oil-water separation and self-assembly. Magnetron sputtering technology appeared in the 1970s as a physical deposition method with the characteristics of high speed and low damage [25]. It can not only improve the manufacturing efficiency and the performance of the SERS substrate, but also control the parameters during the sputtering process, and the operation is relatively simple [26–28]. Rhodamine 6G (R6G) is widely used as a detection molecule [29], and its vibration characteristics and Raman peak distribution are also well known, so we choose R6G for screening the substrate with best performance and testing the performance of the substrate.
Razor clam is a common biological material, which is composed of nacre, prismatic and cuticle from inside to outside. From the scanning electron microscopy images, we can observe that the structure of razor clams with cuticle is very messy, and there is no proper structure that can generate “hot spots”. However, the razor clams, which become a prismatic layer after removed the outermost layer of the cuticle, have a certain columnar structure, albeit with irregular structure. In this work, natural razor clams without cuticle (here collectively referred to as RC) were selected as the substrate material. According to the analysis of our experimental results, the magnetron sputtering time of Ag with the best enhancement effect of the SERS substrate is 5 min (Ag-5/RC). Then, the dense Ag nanoparticles layer was modified on the substrate (AgNPs/Ag-5/RC) by the method of oil-water separation and self-assembly. The modified substrate can greatly reduce the relative standard deviation ($RSD$) (from above 30% to below 20%) and greatly enhance the sensitivity for R6G molecules (from 10−12 M to 10−18 M). The polarization dependence of the substrate has also been improved from polarization-dependent to polarization-independent. The method of depositing dense Ag nanoparticles layer to improve the performance of SERS substrates also provides new ideas for the application of irregular substrates.
2. Experimental section
2.1. Materials and instruments
The natural razor clam (RC) was collected from Repulse Bay in Qinhuangdao and the silver (99.99%) target for magnetron sputtering was purchased from Nanchang Material Technology Co., Ltd. Rhodamine 6G and ethanol absolute were purchased from J&K Scientific LTD. N-Hexane was purchased from Damao Chemical Reagent Factory in Tianjin. Thiram (TMTD) was purchased from AGROLEX Ltd. Silver sol was synthesized by School of Environmental and Chemical Engineering in Yanshan University. Deionized water was obtained from the Key Laboratory of Physics of Microstructure Materials of Hebei Province. The morphology information of the substrate was characterized by scanning electron microscope (SEM, Hitachi S-4800 II). The Raman spectra were measured by Raman system (inVia), while the polarized SERS spectra were collected by alpha300 R-WITec. UV–vis absorption spectra were monitored by Shimadzu UV-2550 system.
2.2 Preparation of substrate
Figure 1 illustrates the preparation process of Ag/RC and AgNPs/Ag/RC substrates and the corresponding SERS detection. The magnetron sputtering process was proceeded at room temperature, and the sputtering parameters were as the follows: voltage at 100 V, the current at 200 mA, and the vacuum at 10−3 pa. The RC were decorated with Ag nanoislands with different sputtering time. The sputtering time were set to 2.5, 5, 10, 15 and 20 min respectively, and they are defined as Ag-2.5/RC, Ag-5/RC, Ag-10/RC, Ag-15/ RC and Ag-20/RC for convenience. R6G of 10−8 M concentration was used as the probe molecule, and the optimal substrate was selected for further study. However, we found that the $RSD$ value of this substrate was high, which was not suitable for the study of surface-enhanced Raman scattering. Hence, we use the Oil-water separation self-assembly method laid dense layer of 55 ± 5 nm Ag nanoparticles on the optimal substrate.
The method of oil-water separation self-assembly as follows: Pour N-Hexane into a clean petri dish, and then slowly drop the pretreatment silver sol (The supernatant was removed by centrifugation and mixed by ultrasound) on the N-Hexane. At this time, the silver sol will be evenly distributed on the upper layer of the N-Hexane. Use a pipette to take an appropriate amount of absolute ethanol and drop it on the mixed interface of silver sol and N-Hexane. Then, Ag nanoparticles will be adsorbed on the interface of N-Hexane, and under the induction of ethanol, the adsorbed Ag nanoparticles will slowly be increasing, and forming a mirror-like Ag film. Using tweezer to place the prepared Ag-5/RC substrate under the Ag film, and then lift it up. Ag film remains on the substrate and forms a dense layer of Ag nanoparticles on its surface. The pulled substrate is placed on a paper sheet, and the upper Ag nanoparticles are naturally dried, and the new substrate (AgNPs/Ag-5/RC) is stored in a vacuum-like environment for subsequent research.
In addition, the samples for detecting thiram (TMTD) on apples are processed as follows: clean the apples with deionized water, then cut them into small pieces (1 cm × 1 cm). Use a pipette to drop 10 µl of the pre-prepared TMTD solution of different concentrations (5×10−3 mg/ml, 2×10−2 mg/ml and 5×10−2 mg/ml) on the skin of the apple of the specified size, and then naturally dry. After drying, the solid AgNPs/Ag-5/RC substrate was pressed 10 s on the apple skin to obtain the AgNPs/Ag-5/RC substrate with TMTD analyte for detection.
2.3 SERS measurements
Whether there are Ag nanoislands on the surface of the RC was checked by SEM. R6G is prepared in deionized water with a concentration of 10−3 M ∼ 10−18 M. In order to screen out the best substrate, 10−8 M R6G droplets were added to the previously prepared substrates with different Ag sputtering time. Using a 532 nm laser can avoid fluorescence interference in order to achieve SERS enhancement. All the Raman signals are measured at room temperature with 532 nm laser and the incident power is 0.05 mW. All polarization studies of SERS substrates use 532 nm laser as the excitation source.
3. Results and discussion
3.1. Morphology characterization
The SEM images (Fig. 2) show the surface morphs with different sputtering time on RC. Figure 2(a1) shows RC with the outermost cuticle, and Figs. 2(a)–2(d) show that without the cuticle. By removing the outermost cuticle, the prismatic layer is exposed on the outermost, and the irregular columnar structure appears, although it is more regular than the cuticle. Figures 2(a)–2(d) shows the top-views of RC, Ag-2.5/RC, Ag-5/RC, and Ag-10/RC with the top diameter of 60 ∼ 240 nm, respectively. Because most of the RCs are dense, irregularly distributed, even overlapping, almost all of the Ag nanoislands grow on columnar structure. As shown in Fig. 2(c), the top diameter with Ag nanoislands are larger in size and more evenly distributed, and a large number of the “hot spots” can generated between neighboring Ag nanoislands. With the increase of sputtering time, the top diameter of modified nanoislands are increased and the distance between adjacent modified nanoislands is decreased. The Ag-5/RC shows large density of Ag nanoislands and good uniformity, which probably induce better Raman enhancement. With a long-time sputtering, the gaps between nanoislands were almost filled with Ag, leading to a decrease in the density of “hot spots” between adjacent nanoislands. At the same time, Raman enhancement is also significantly reduced.
Fig. 2. (a1) is the SEM image with the outermost cuticle. (a-d) Top-view SEM images of RC, Ag-2.5/RC, Ag-5/RC and Ag-10/RC, respectively.
3.2. SERS performances
UV-vis absorption spectrum is used to select the excitation light wavelength which is suitable for the substrate with the best SERS enhancement. Figure 3 shows the UV-vis absorption spectra of Ag-5/RC, AgNPs/Ag-5/RC and R6G respectively. Among them, the black line represents the Ag-5/RC substrate, and its absorption peak center is around 310 nm. Due to the limitations of our experimental equipment, currently there are only excitation wavelengths of 532 nm, 633 nm and 785 nm. If these lasers are used for excitation, the plasmon resonance effect is not obvious, and the enhancement effect is not ideal. After the dense Ag nanoparticles were modified by the oil-water separation self-assembly method, the UV-vis absorption spectrum of the new substrate (AgNPs/Ag-5/RC) is shown by the red line. The absorption peaks mainly exist in the two wavebands around 300 nm and 500 nm. Compared with Ag-5/RC, the increased waveband confirms the presence of Ag nanoparticles. If 532 nm laser is used to irradiate the substrate, there will be a more obvious plasmon resonance effect. At the same time, more gaps will be generated between particles and between particles and the Ag film, which can significantly improve the enhancement effect. We also tested the absorption spectrum of R6G (blue line) and found that it has a wider band with many obvious absorption peaks before 650 nm. If R6G is irradiated with a 532 nm laser, the measurement result will be ideal. Therefore, compared with other lasers, 532 nm laser is more suitable for subsequent measurement.
Figure 4(a) shows the measured SERS spectra on the substrates (Ag-2.5/RC, Ag-5/RC and Ag-10/RC, Ag-20/RC) with different sputtering time for forming Ag nanoislands. The intensities of SERS spectra are quite different. Figure 4(b) shows a zoom-in image of the characteristic peak at 1360 cm-1 in Fig. 4(a). It can be obviously observed in Fig. 4(b) that Ag-5/RC had the strongest SERS enhancement compared with other SERS-active substrates. The common Raman characteristic peaks of R6G molecule is 613, 772, 1126 cm-1, representing in-plane bending of C-C-C ring, and out-plane, in-plane bending vibration of C-H. The characteristic peaks of 1190, 1309, 1360, 1509, 1572 and 1649 cm-1 are due to the tensile vibration mode within the aromatic C-C plane. [30–34]. As shown in Fig. 4(c), the SERS spectrum strength changed with the concentration of R6G (from 10−7 M to 10−12 M), and the characteristic peak of R6G could be observed obviously at the concentration of 10−12 M. Figure 4(d) shows the Raman spectrum of 10−8 M R6G which were measured at 20 randomly selected locations from the Ag-5/RC substrate to illustrate the reproducibility of the SERS-active substrates. The spectra and the Raman intensity with obvious changes indicate that the reproducibility of the substrate-to-substrate is weak. Figure 4(e) and Fig. 4(f) show the $RSD$ at the characteristic peaks of 772 cm-1 and 1360 cm-1 respectively. The $RSD$ is calculated by the following formula [35]:
The number of Raman spectra is n=20, ${I_i}$ is the Raman signal intensity of each characteristic peak, and the average intensity of the Raman signal is $\bar{I}$. The $RSD$ of 772 cm-1 characteristic peak is 30.81%, and the $RSD$ of 1360 cm-1 characteristic peak is 24.23%, both of which are larger than 20%. These results indicate that the Ag-5/RC SERS active substrate has poor reproducibility and uniformity, related to the irregular structure of the RC.
Fig. 4. (a) SERS spectra of 10−8 M R6G on SERS substrates with different sputtering times (Ag-2.5/RC, Ag-5/RC and Ag-10/RC, Ag-20/RC). (b) Comparison of the Raman intensities of different SERS-active substrates at the characteristic peak at 1360 cm-1. (c) SERS spectra of R6G for different concentrations on Ag-5/RC. (d) SERS spectra of R6G at the concentration of 10−8 M on 20 randomly selected positions of the Ag-5/RC. (e)-(f) the main Raman intensities of 10−8 M R6G at characteristic Raman peaks (e: 772 cm-1, f: 1360 cm-1) and the corresponding $RSD$ values.
The reproducibility of the SERS substrate is an important factor in the practical application of SERS [36]. It is generally believed that the Raman reproducibility of the SERS platform for quantitative detection should satisfy the RSD value of less than 20% [37]. In order to improve the reproducibility of SERS active substrates, we used the oil-water interface self-assembly method [38,39] to add a dense Ag nanoparticles layer on Ag-5/RC. Figure 5(a) is the SEM image of the new SERS-active substrates (AgNPs/Ag-5/RC). From the image, we can see that the Ag nanoparticles are uniformly attached to the substrates, making the surface of the substrate more uniform compared with the previous Fig. 2(c). Figure 5(a1) is the SEM image of Ag nanoparticles with a size of 55 ± 5 nm. As shown in Fig. 5(b), the SERS spectrum strength changed with the concentration of R6G (from 10−8 M to 10−18 M), the characteristic peak could be observed obviously even at the concentration of 10−18 M. This ultra-sensitive characteristic also will make AgNPs/Ag-5/RC have a good performance in application detection. Figure 5(c) is a point-by-point SERS mapping, 5 × 5 µm2 is randomly selected (step size is 1 µm), and the brightness of each grid represents the intensity of the corresponding SERS signal. The relatively uniform color distribution in the figure shows that the intensity of the Raman signal is relatively uniform at different test positions. Figure 5(d) shows the Raman signal intensities of 10−8 M R6G which were measured at 20 randomly selected locations from the AgNPs/Ag-5/RC substrates to illustrate the reproducibility of the SERS-active substrates. Compared with Fig. 4(d), the peak intensity in Fig. 5(d) is relatively uniform, indicating that the substrate with Ag nanoislands has better substrate-to-substrate reproducibility. We also use formula (1) to calculate the $RSD$ at the characteristic peaks of 772 cm-1 and 1360 cm-1 as shown in Fig. 5(e) and Fig. 5(f). Compared with the previous results in Fig. 4(e) and Fig. 4(f), the $RSD$ values of these two characteristic peaks dropped from 30.81% to 18.90%, and from 24.23% to 19.25%, respectively. The addition of Ag nanoparticles makes the previously substrate more uniform and smooth, and generate more “hot spots”. Hence, the new substrate has been improved in terms of uniformity, reproducibility, and enhanced performance.
Fig. 5. (a) Top-view SEM of AgNPs/Ag-5/RC. (a1) Top enlarged SEM of AgNPs. (b) SERS spectra of R6G for different concentrations on AgNPs/Ag-5/RC. (c) SERS mapping (5${\times} $5 $\mathrm{\mu}$m2, 1 $\mathrm{\mu}$m step size) of the characteristic peak of R6G at 1360 cm-1 on AgNPs/Ag-5/RC. (d) SERS spectra of R6G at the concentrate of 10−8 M on 20 randomly selected positions of the AgNPs/Ag-5/RC. (e)-(f) the main Raman intensities of 10−8 M R6G at characteristic Raman peaks (e: 772 cm-1, f: 1360 cm-1) and the corresponding $RSD{\; }$value.
3.3. Polarization property study of the Ag-5/RC substrate and AgNPs/Ag-5/RC substrate
Polarization dependence is an important issue in the study of SERS substrates. For example, the same nano-structural substrate irradiated with different excited polarized light will produce different SERS effects, and the different nano-structural substrates irradiated with the same excited polarized light will also have different SERS effects [40]. Therefore, we use Ag-5/RC and AgNPs/Ag-5/RC substrates to study the relevant polarization characteristics. Because the polarization instrument is different from the previous Raman instrument, some of their inherent parameters are different, resulting in significant changes in the intensity of the Raman signal. Figure 6(a) is a schematic diagram of the polarization measurement on the AgNPs/Ag-5/RC substrate. Through the rotation of polarization directions, Raman spectra measured at different polarization angles ($\mathrm{\theta }$) are obtained to explore the property of polarization dependence. The polarization measurement was also carried out on the Ag-5/RC substrate. Figure 6(b) is a schematic diagram of the Raman intensities of the two substrates as a function of the polarization angle for the peak of 1361 cm-1. The yellow line represents the Ag-5/RC, and the blue line represents the AgNPs/Ag-5/RC. For the AgNPs/Ag-5/RC, the Raman intensity does not change significantly with the polarization angle, in Fig. 6(b), most of the points are located in the blue circular area, indicating that the AgNPs/Ag-5/RC has a good uniformity performance. However, for Ag-5/RC, the Raman intensity is low and greatly affected by polarization angle. This also confirms that the modification of Ag nanoparticles can improve the uniformity of the substrate. Figure 6(c) and Fig. 6(d) show the 1361 cm-1 peak intensities of 10−8 M R6G at different polarization angles on Ag-5/RC and AgNPs/Ag-5/RC substrates, respectively. After the modification of Ag nanoparticles, the $RSD$ value dropped from 34.78% to 14.53%, which further confirms that the polarization-independent performance of AgNPs/Ag/RC was more and more remarkable.
Fig. 6. (a) The schematic diagram of polarization measurement of AgNPs/Ag-5/RC SERS substrate. (b) The schematic diagram of the relationship between the polarization angle and the Raman intensity. The yellow line represents the Ag-5/RC substrate, and the blue line represents the AgNPs /Ag-5/RC substrate. (c) and (d) show the SERS signal intensity distribution of the characteristic peak of 1361 cm-1 at the R6G concentration of 10−8 M on Ag-5/RC and AgNPs/Ag-5/RC substrates.
3.4. Calculation of SERS enhancement factor ($\; EF$)
SERS is mainly due to the enhancement of local electromagnetic fields, and the $EF$ is an important parameter to characterize the performance of SERS. The $EF$ is usually calculated by the intensities of the absorbed probe molecules on the SERS active substrate, horizontal glass or silicon wafer. Figure 7 shows the Raman spectrum of 10−3 M R6G on a blank SiO2 (red line) and the SERS spectrum of 10−8 M R6G on AgNPs/Ag-5/RC substrate (black line). The SERS $EF$ of the AgNPs/Ag-5/RC substrate is calculated by the following formula [41,42]:
Fig. 7. The SERS spectrum of 10−8 M R6G dropped on AgNPs/Ag-5/RC substrate, and the Raman spectrum of 10−3 M R6G dropped on blank SiO2.
${N_A}{\; }$is the Avogadro constant, $M{\; }$is the concentration of the R6G solution, ${V_{solusiton}}$ is the volume of the droplet, ${S_{sub}}$ is the area of the R6G solution on the different substrates, and ${S_{laser}}$ is the area of the laser spot. In this experiment, the area of R6G molecules on SiO2 is about 1.2-time larger than that on the SERS substrate. The diameter of the laser spot is 1µm, so the area of the laser spot is about 0.785 µm2. Therefore, the radio of ${\raise0.7ex\hbox{${{N_{Raman}}}$} \!\mathord{\left/ {\vphantom {{{N_{Raman}}} {{N_{SERS}}}}} \right.}\!\lower0.7ex\hbox{${{N_{SERS}}}$}}$ was calculated to 8.33${\times} $104. According to the above data, the $EF{\; }$of AgNPs/Ag-5/RC substrate is 5.45${\times} $106. In addition, the $EF{\; }$of Raman peaks at other positions of R6G are also calculated in Table 1. The oil-water separation self-assembly method is applied to the messy substrate, and the experimental results show that the substrate modified by Ag nanoparticles have good SERS performance.
Table 1. The enhancement factor calculated from different Raman peaks of R6G on AgNPs/Ag-5/RC substrate.
3.5. 3D finite-different time-domain simulation
3D finite-difference time-domain simulation (3D-FDTD) is a simulation method commonly used to analyze the spatial distribution of local electric fields on nano-rough surfaces of noble metals [43]. The main object of the study is the substrate structure, while, probe molecules are not considered in the simulation. In order to ensure the reliability of the simulation results, the various parameters of Ag in this article refer to Gai et al.'s theoretical research on the wideband modified Drude model [44]. Figure 8(a) is a structural diagram of the AgNPs/Ag-5/RC substrate, in which Ag nanoparticles are modified on the Ag nanoislands, and Fig. 8(b) is the calculated planes. Besides, the diameter of each Ag nanoislands is 140 nm, and the diameter of the Ag nanoparticles is 50 nm (size data obtained from the SEM image in Fig. 2). The wavelength of the incident laser is 532 nm, the K direction is perpendicular to the surface, and the polarization direction of the laser is E direction. Figure 8(c) and Fig. 8(d) are the spatial distribution of the electric-field intensity in x-y plane and x-z plane shown in Fig. 8(b), respectively. It can be clearly seen from the figure that the main “hot spots” (“hot spots I”) exist between the Ag nanoislands. Compared with the electric field intensity of “hot spots I”, the intensity of “hot spots II” is relatively weak. In the process of simulation, the main research is to study the electromagnetic enhancement, which focus on the surface morphology, structure model and specific size of Ag nanoislands and Ag nanoparticles modified on SERS substrates [25]. From the relationship between the Raman enhancement scales and the local field [45], we can get
Parameter ${E_{loc}}({{\omega_{exc}}} )$ and parameter ${E_{inc}}({{\omega_{exc}}} )$ are E and E0 in FDTD respectively. The maximum electric field calculated by the simulation is about ∼ 36.2 V·m-1. The calculated theoretical${\; }EF{\; }$1.7×106 is close to the experimental value (2.04∼5.53) × 106.
Fig. 8. (a) The FDTD model of the AgNPs/Ag-5/RC substrate. (b)The calculated planes. (c) and (d) are the spatial distribution of the electric field intensity in the x-y and x-z planes defined in (b), respectively.
3.6 Detection of thiram (TMTD) by the AgNPs/Ag-5/RC substrate
Thiram (TMTD) is a kind of fungicide, which is widely used in plant sterilization, preservation of ripe fruits and storage and transportation of vegetables. However, excessive use of TMTD can lead to soil pollution, seepage into groundwater, and TMTD ingested from fruits and vegetables will also have difficulty breathing [46]. It is necessary to detection TMTD in the practical application of SERS.
Therefore, the AgNPs/Ag-5/RC substrate was employed for the detection of TMTD on the apple skin. Typically, 10 µL of diverse concentrations of as-prepared TMTD solutions were dropped on the apple and dried in air. After drying, the solid AgNPs/Ag-5/RC substrate was pressed on the apple skin to obtain the AgNPs/Ag-5/RC substrate with TMTD for detection. Figure 9(a) shows the spectra of different concentrations of TMTD on AgNPs/Ag-5/RC substrate. Among them, the concentration of the black line, red line and blue line are 500 ng/cm2, 200 ng/cm2, 50 ng/cm2, respectively. The main characteristic peaks of TMTD exist at 563, 1147, 1383 and 1516 cm-1, which are SS stretching, C-N stretching and CH3 rock, C-N stretching and CH3 deformation, CN stretching and CH3 deformation, respectively [47,48]. Figure 9(b) shows the Raman signal intensities of 500 ng/cm2 TMTD from apples which were measured at 20 randomly selected locations from the AgNPs/Ag-5/RC substrate. Meanwhile, the RSD (n = 25) of 16.58% and 13.43% were calculated from the Fig. 9(c) and Fig. 9(d), which demonstrated the relatively high reproducibility of AgNPs/Ag-5/RC substrate. The clear spectrum at a concentration of 50 ng/cm2 indicated that the AgNPs/Ag-5/RC substrate has potential in TMTD detection, and it is possible for the substrate modified by Ag nanoparticles to be used in practical applications.
Fig. 9. (a) AgNPs /Ag-5/RC substrate for the detection of different concentrations of TMTD on Apple. (b) SERS spectra of TMTD at the concentrate of 500 ng/cm2 on 20 randomly selected positions of the AgNPs/Ag-5/RC. (c) - (d) the main Raman intensities of 500 ng/cm2 TMTD at characteristic Raman peaks (c: 562 cm-1, d: 1381 cm-1) and the corresponding $RSD$ value.
4. Conclusion
In summary, the method of modifying a dense Ag nanoparticles layer on the surface of the substrate through oil-water separation and self-assembly method has significantly improved the SERS signal, polarization dependence, and reproducibility of the substrate. The natural razor clam material without cuticle has irregular columnar structure, leading to a high $RSD$ value, nonuniformity and polarization-dependent. After being modified with Ag nanoparticles, the above-mentioned undesirable optical properties can be greatly improved. The 3D-FDTD method was employed to simulate the modified substrate, and the results demonstrated that the main “hot spots” were concentrated between the Ag nanoislands, and the secondary “hot spots” are among the adjacent Ag nanoparticles. The strategy that modified with Ag nanoparticles layer provides a new idea to improve the SERS performance of irregular substrates.
Funding
Science and Technology Project of Hebei Education Department (ZD2019069); National Natural Science Foundation of China (11674275, 21872119, 22072127).
Disclosures
The authors declare no conflicts of interest.
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