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Abstract
The combination of new noble metal nanomaterials and surface enhanced Raman scattering (SERS) technology has become a new strategy to solve the problem of low sensitivity in the detection of traditional Chinese medicine. In this work, taking natural cicada wing (C.w.) as a template, by optimizing the magnetron sputtering experimental parameters for the growth of Ag nanoparticles (NPs) on vanadium-titanium (V-Ti) nanorods, the nanogaps between the nanorods were effectively regulated and the Raman signal intensity of the Ag15/V-Ti20/C.w. substrate was improved. The proposed homogeneous nanostructure exhibited high SERS activity through the synergistic effect of the electromagnetic enhancement mechanism at the nanogaps between the Ag NPs modified V-Ti nanorods. The analytical enhancement factor (AEF) value was as high as 1.819 × 108, and the limit of detection (LOD) was 1 × 10−11 M for R6G. The large-scale distribution of regular electromagnetic enhancement “hot spots” ensured the good reproducibility with the relative standard deviation (RSD) value less than 7.31%. More importantly, the active compound of Artemisinin corresponded the pharmacological effect of Artemisia annua was screened out by SERS technology, and achieved a LOD of 0.01 mg/l. This reliable preparation technology was practically applicable to produce SERS-active substrates in detection of pharmacodynamic substance in traditional Chinese medicine.
© 2022 Optica Publishing Group under the terms of the Optica Open Access Publishing Agreement
1. Introduction
With the continuous optimization of the preparation process of noble metal nanostructures and the improvement of noble metal optical theory, high sensitivity detection technology has shown a great potential in molecular level detection [1,2]. As a new medical detection method, SERS technology can provide rich spectral and structural information of samples, and has become a research “hot spot” in the fields of molecular spectroscopy and biomedical sensing [3]. SERS technology pointed out that when probe molecules adsorbed on the surface of rough noble metal nanostructures through chemical bonds and other effects, the Raman signal intensity of probe molecules can be enhanced by 106−1015 times [4]. The discovery of SERS effect has completely solved the key problems of weak Raman signal intensity, low sensitivity and easy to be interfered by fluorescence in trace detection, and has become one of the most potential trace substance detection tools in the fields of material science, environment monitoring and biomedical sensing [5–8].
The improvement of SERS theory has brought a revolutionary new method to the field of biomedical sensing [9–11]. SERS technology, due to its high sensitivity, allows people to adjust the molecular sensitivity in the amolar range and reach the single-molecule detection level in specific circumstances [12]. Due to the high inspection speed and the richness of information content, the Raman “fingerprint” of biomolecules obtained by the label free SERS sensor can provide insight into their conformation and structure, and has great application prospects in the detection of biological structures such as proteins and the preliminary identification of cancers [13]. Sung Gyu Park et al [14]. proposed in-situ surface modification of Au nanopillar electrodes by electrodeposition. New plasmonic nanostructures were constructed on the Au nanoarrays, which can effectively interfer with the biomolecules. This method can reliably detect ascorbic acid, dopamine and uric acid without label. The LOD values were 1 nmol/l, 0.1 nmol/l and 1 nmol/l, respectively. Professor Feng’s team of Fujian Normal University [15] combined SERS technology with surface molecularly imprinted polymer technology. They embedded a stable internal standard meter in the gap of core-shell nanostructures and constructed Au@Ag core-shell nanostructure. This Au@Ag core-shell SERS sensor was used to quantitatively detect carcinoma embryonic antigen which were closely related to a variety of cancers. When detecting carcinoma embryonic antigen in clinical serum samples, it was found that the limit detection range of this method was larger than that of electrochemiluminescence immunoassay, and the required sample volume was smaller. Shandong Normal University and Liaocheng University jointly developed a series of MoS2-based [16] and graphene-based heterostructures with novel morphology, such as Ag NP/graphene layers [17], MoS2-NPs/CeO2-NPs [18], hat-shaped MoS2 [19] and MoS2/Au/Ag [20] substrates. These substrates exhibited highly enhanced SERS performance, while realizing highly sensitive detection of thiram, alanine aminotransferase, p-nitrothiophenol and p’-dimercaptobenzene. Other research group also developed silicon plasmonic fiber tip biosensor [21], hazardous material sensor [22] and high-conductive nanowires electrodes sensor [23] to expected to realize high-performance sensing in SERS field.
In recent years, researchers often use thin-layer chromatography to identify the pharmacodynamic substance of traditional Chinese medicine [24–26]. Although this method is accurate for the analysis of traditional Chinese medicine, they can not be widely used because of the strict separation requirements and the long time-consuming analysis process. Raman scattering technology has been proved to be a fast and non-destructive tool to identify traditional Chinese medicine [27]. For example, ginseng and its counterfeit products were successfully identified using this technology [28]. However, in the previous Raman spectroscopy studies, only qualitative identification of the pharmacodynamic substance of traditional Chinese medicine can be done, without quantitative component information. This was due to the low efficiency of Raman scattering itself, and the content of pharmacodynamic substance in traditional Chinese medicine was relatively low (the content of pharmacodynamic substance in different traditional Chinese medicine was different, usually below 10%) [29].
At present, according to the research status at home and abroad, few study on the pharmacodynamic substance of traditional Chinese medicine using SERS technology reported. In fact, SERS detection technology has the advantages of fast detection speed, simple operation, strong stability, good reproducibility, high sensitivity and no need to separate and extract samples [30,31]. It is very suitable for the trace detection and quality evaluation analysis of traditional Chinese medicine.
Combined with the above SERS substrates preparation technology and SERS application, this paper proposed a method to prepare multi-stage composite SERS substrate with high performance using V-Ti-based material as template by magnetron sputtering technology and physical deposition method. The schematic diagram of the Ag/V-Ti/C.w. preparation process was exhibited in Fig. 1. Firstly, the regular V-Ti nanorod array was fabricated by magnetron sputtering using the regular nanorod structure on the surface of C.w. as a template. After the surface of C.w. was modified with V-Ti nanomaterial, the distribution range of electromagnetic enhancement “hot spots” of plasmonic nanostructures could be greatly increased, and the statistical result of ultra-sensitive detection could be true and reliable [32]. On the other hand, the widely sourced V-Ti material was used as the template of the SERS detection platform, which effectively reduced the preparation cost of the SERS substrate and simplified the process. Secondly, Ag NPs were modified on the V-Ti/C.w. template by magnetron sputtering to construct Ag15/V-Ti20/C.w. nanostructures. There were many nanogaps about 10 nm between the nanorods of the Ag15/V-Ti20/C.w. nanostructure, which could excited multi-stage electromagnetic enhancement “hot spots” and further improved the Raman scattering performance of the Ag15/V-Ti20/C.w. composite nanostructures. Thirdly, We physically deposited 30 nm Au NPs on the surface of V-Ti/C.w. to prepare Au NPs30/V-Ti20/C.w. substrate. Its Raman signal enhancement ability was much less than that of Ag15/V-Ti20/C.w. substrate. Therefore, the Ag15/V-Ti20/C.w. substrate was employed to detect R6G, and the LOD was located at 1 × 10−11 M. This novel Ag15/V-Ti20/C.w. nanoarray with substantial “hot spots” exhibited high SERS signal reproducibility, with the RSD value less than 7.31%. Meanwhile, the regular V-Ti nanorods provided a larger available area for loading Ag nanomaterials with SERS activity, and made polarization independence possible. More importantly, The Ag15/V-Ti20/C.w. substrate combined with Raman enhanced spectroscopy technology was used to realize the rapid, accurate and quantitative trace detection of Artemisinin, and the LOD was 0.01 mg/l. The experimental results provide a new way for the test of various pharmacodynamic substance of traditional Chinese medicine, and provide theoretical and experimental support for improving the new strategies for the testing of pharmacodynamic substance of other traditional Chinese medicine.
Fig. 1. Schematic illustration of the fabrication process of the Ag15/V-Ti20/C.w. and Au NPs30/V-Ti20/C.w. nanoarray and the SERS measurement by the Raman system.
2. Experimental section
In the experiment section, we first introduce the information of chemicals and materials required for the experiment. Then, according to the technical route of the experiment, the preparation process of SERS substrate was described. Next, the adsorption process between different substances to be measured and SERS substrate was described. Finally, the morphology of SERS substrate and the characterization process of SERS properties were described.
2.1 Chemicals and materials
The Ag target (diameter: 60.0 mm, thickness: 2.0 mm, purity: 99.99%) and V-Ti target (diameter: 60.0 mm, thickness: 2.0 mm, purity: 99.99%) were purchased from Nanchang Hanchen New Materials Technology Co., Ltd., Nanchang, China. The C.w.s were obtained from Beijing Jiaying Grand Life Sciences Co., Ltd. R6G was obtained from J&K Scientific Ltd., Beijing, China. Artemisinin (CAS Number: 63968-64-9) was purchased from BASF Biotechnology Co., Ltd. Hefei, China. AuCl3·HCl·4H2O (99.8%) and Na3C6H5O7·2H2O (99.9%) were purchased from Aladdin, Shanghai, China. Deionized water (18.25 MΩ) was used to prepare the solutions throughout the experiment.
2.2 Preparation of Au NPs
In this experiment, the sodium citrate reduction method was applied to prepare Au NPs according to the previous work [33]. Before the experiment, all the glass instruments used were fully soaked in chloroazotic acid, then washed 3 times with deionized water and dried before used. During the experiment, firstly, poured 50 ml deionized water into the round-bottom flask, then 0.5 ml AuCl3·HCl·4H2O (99.8%) aqueous solution (1%) was dropped into the round-bottom flask with a pipette, and added a magnetic stirrer to stir. Second, when the solution was completely boiling, 1.6 ml of Na3C6H5O7·2H2O (1%) was quickly dropped. After 20 min, the color of the solution turned wine red, then stopped heating and cooled naturally to room temperature. Last, the AuNPs solution was concentrated with centrifuge for 10 min with a centrifugal rate of 15000 rpm.
2.3 Preparation of SERS substrates
The V-Ti/C.w. nanorods and Ag/V-Ti/C.w. nanorods were fabricated by the high vacuum magnetron sputtering and an ion beam composite thin film deposition system (FJL560, Shenyang Scientific Instruments Co., LTD., Shenyang, China) according to the previous work [34]. Firstly, the V-Ti target was fixed on the sputtering target. Secondly, the C.w. nanotemplates were fixed on the sample table in the sputtering chamber. Then, when the mechanical pump and molecular pump were all turned on, the pressure in the sputtering chamber reached 3.5 × 10−3 Pa after about 20 min. At this time, turned on the flow indicator and controlled the argon flow at 120 ml/m. Finally, when the magnetron sputtering power was set to 150 W, the V-Ti target was successfully lit up and the V-Ti coating began. The preparation process of Ag/V-Ti/C.w. substrate was the same as above, the only difference was that the argon flow was 100 ml/m and the sputtering power was 100 W. For convenience, we defined the terminologies beforehand that described different substrates. The V-Ti/C.w. substrates fabricated at the sputtering time of x min was defined as V-Tix/C.w.. With one accord, the Agy/V-Tix/C.w. referred to that the sputtering time of Ag was y min and the sputtering time of V-Ti was x min.
The prepared AuNPs was decorated on the surface of V-Ti/C.w. by a simple physical deposition and dried in a vacuum drying oven for 10 min at the temperature of 50 °C, and the Au NPs/V-Ti/C.w. substrate was fabricated.
2.4 Probe molecules adsorption
R6G solutions with different concentrations were prepared by 10 times dilution method with deionized water. For each substrate, 10 µl R6G (1 × 10−3 to 1 × 10−11 M) was added onto the prepared substrates and dried at room temperature to achieve the adsorption of as many R6G molecules as possible on the surface of the prepared substrates. Similarly, Artemisinin solutions of different concentrations were also prepared by 10 times dilution method. Because the solvent was glacial acetic acid, when 10 µl artemisinin solution dropped on the surface of the substrate, for the cohesive force was less than the adhesive force, no liquid drops can be formed on the surface of the substrates. Therefore, we soaked the substrate in Artemisinin solutions with different concentrations for 2 h, and then took it out and dried it naturally for SERS measurement.
2.5 Characterization
The field emission scanning electron microscopy (FE-SEM) (SU8220, Hitachi, Tokyo, Japan) and atomic force microscopy (AFM) (Bruker Dimension ICON, Germany) were employed to characterized the morphology of V-Tix/C.w., Agy/V-Tix/C.w. and Au NPs/V-Tix/C.w. substrates. The morphology of Au NPs was characterized by transmission electron microscope (TEM) (HT7700, High-Technologies Corp., Ibaraki, Japan). UV-vis absorption spectra were recorded by the Shimadzu UV-2550 system (Shimadzu (China) Co., Ltd., Shanghai, China).
SERS spectra were obtained by the microscopic confocal Raman spectrometer (DXR2xi, Thermo Fisher Inc., USA) with a ×50 objective lens. During the SERS measurement, the recorded range was 500 cm−1−2000cm−1, the excitation wavelength was 532 nm, the acquisition time of each Raman spectrum was 10 s and the laser power was set at 5 mW. Polarization-dependent Raman spectra were recorded by a Confocal microscopic Raman spectrometer (Horiba HR Evolution, Horiba Scientific, France).
3. Results and discussion
In this section, we firstly characterized the morphology characteristics of different substrates by SEM, AFM and TEM, and revealed the change rule of nanogap through the morphology characteristics. Secondly, R6G was used as the probe molecule to screen the substrate with the best SERS performance. Finally, the substrate with the best Raman signal response was tested with the Raman signal reproducibility, polarization, sensitivity, AEF value and practicability.
3.1. Morphology characterization of the C.w. and V-Tix/C.w. nanotemplates
Figure 2(a) and (a1) show the FE-SEM images of the the C.w. surface from the top view and the sloping view. We can clearly observe that there were a large scale of rod-like nanostructures existed with a high degree of regularity. The average diameter of the top of the nanorods was 70 ± 3 nm, and the height was approximately 200 ± 5 nm. We have counted the nanogap between 80 nanorods in Fig. 2(a). From Fig. 2(b), we can see that the average size of the nanogap between nanorods was 80 ± 2 nm.
Fig. 2. FE-SEM images of C.w. from (a) top view and (a1) sloping view; FE-SEM images of V-Ti15/C.w. nanorods (c) and V-Ti20/C.w. nanorods (e); The frequency distribution histograms of C.w. nanogaps (b), V-Ti15/C.w. nanogaps (d) and V-Ti20/C.w. nanogaps (f).
It is generally known that the nanogap was one of the key factors that determined the intensity of electromagnetic field [35]. The nanogap with an average size of 80 ± 2 nm was not conducive to the formation of localized surface plasmon resonance (LSPR) effect. Therefore, V-Ti materials from a wide range of sources were modified on the nanorod-like surface to construct V-Tix/C.w. nanotemplates with different morphologies. As shown in Fig. 2(c) and (e), V-Tix/C.w. nanoarrays were obtained by adjusting the sputtering time while keeping other conditions being equal. When sputtering time was set to be 15 min, a great quantity of V-Ti nanorods with an average diameter of 154 ± 5 nm were obtained on the V-Ti15/C.w. nanoarrays. Meanwhile, the V-Ti nanorods still regularly arranged. The frequency distribution histogram was presented in Fig. 2(d), the size distribution of nanogaps obeyed the Gaussian distribution with the peak position centered at 43 ± 2 nm by counting 100 randomly selected samples. Continued to employ magnetron sputtering technology to regulate the top diameter and nanogap of V-Ti nanorods. When the sputtering time was increased to 20 min, as shown in Fig. 2(e) and (f), the top diameter of the V-Ti20/C.w. nanorods was 192 ± 5 nm, and the average size of the nanogap was 20 ± 2 nm. These results confirmed that the magnetron sputtering technology was a powerful tool to regulate the nanomorphology and nanogap. Next, we will modify the plasmonic noble metal nanomaterial on the surface of the V-Tix/C.w. nanorods and continue to regulate the nanogaps of the nanorods, in order to find the optimal V-Ti-based SERS substrate with the strongest LSPR effect.
3.2. Morphology characterization of the Ag/V-Tix/C.w. nanoarray
Among all the plasmonic noble metal materials, Au and Ag were the most popular SERS detection platforms because their LSPR effects fall in the visible region [36,37]. Due to the unique electromagnetic and adjustable LSPR effect, in this paper, Ag nanomaterial was sputtered on the surface of V-Ti based nanotemplates to construct Ag/V-Tix/C.w. nanoarrays. By strictly controlling the sputtering time of Ag nanomaterials and V-Ti nanomaterials, V-Ti nanorod arrays modified by Ag NPs with different morphologies were prepared by magnetron sputtering. The values of special markers such as the top diameter of nanorods and nanogaps were calculated statistically.
As shown in Fig. 3(a), when the sputtering time of Ag NPs was 10 min, the Ag10/V-Ti15/C.w. nanoarrays obtained a rough cauliflower-like nanomorphology with an average top diameter of 188 ± 5 nm. The side-view FE-SEM image of Ag10/V-Ti15/C.w. nanoarrays was presented in Fig. 3(b). Compared with the nanogap size of 43 ± 2 nm in V-Ti15/C.w. in Fig. 2(c), the nanogap size of Ag10/V-Ti15/C.w. substrate was reduced to 35 ± 2 nm. The results demonstrated that magnetron sputtering was a practical and excellent technical method to control the size of nanogap. When the magnetron sputtering time of the Ag NPs was continuously adjusted to 15 min, the Ag15/V-Ti20/C.w. nanoarray was obtained, as shown in Fig. 3(c), the rough cauliflower-like nanomorphology on the top of the nanorods disappeared, which was replaced by regular spherical nanomorphology with an average diameter of 200 ± 5 nm. Meanwhile, the side-view FE-SEM image of Ag15/V-Ti20/C.w. substrate was exhibited in Fig. 3(d1). There were a large range of 10 nm and sub-10 nm nanogaps existed on the surface of Ag15/V-Ti20/C.w. nanoarray. This result indicated that abundant suitable nanogaps on Ag15/V-Ti20/C.w. nanoarray could inspired a great deal of electromagnetic enhancement “hot spots” and further enhanced the scattering cross-section, which was related to a red-shifting of the dipole plasmon wavelength as the gap size was decreased [38]. Seen from the side-view FE-SEM image of Ag15/V-Ti20/C.w. in Fig. 3(d1), there were two types of electromagnetic enhancement “hot spots” distributed on the whole substrate. Type-1 was formed on the top of Ag15/V-Ti20/C.w. nanorods for the existence sub-10 nm nanogaps. Type-2 was generated between the neighboring Ag nanorods. Due to the regular distribution of the Ag15/V-Ti20/C.w. nanorods, the whole substrate had more opportunities to inspire the LSPR effect under incident light, thus, enhancing the intensity of Raman scattering signal. Figure 3(d2) exhibited the AFM image of Ag15/V-Ti20/C.w. nanorods which was in keeping with the FE-SEM image with an average height of 500 ± 5 nm.
Fig. 3. FE-SEM images of Ag10/V-Ti15/C.w. from (a) top view and (b) side view; FE-SEM images of Ag15/V-Ti20/C.w. from (c) top view and (d) side view; Energy dispersive spectroscopy images of Ti (e), V (f) and Ag (g) elements.
The uniform distribution of Ag and V-Ti on the surface of nanorods was observed and was further confirmed by the energy dispersive spectroscopy images of Ti, V and Ag elements in Fig. 3(e)–(g). A large amount of closely adjacent Ag NPs favored the generation of powerful electromagnetic enhancement “hot spots” to enhance the Raman signal intensity. In addition, the uniform distribution of Ag and V-Ti nanomaterials on the nanorods of the C.w. were conducive to improving the Raman signal reproducibility of the Ag15/V-Ti20/C.w. SERS substrate.
3.3 Morphology characterization of the Au NPs30/V-Ti20/C.w. nanoarray
In this paper, in order to continuously regulate the influence of nanogap on the SERS performance of noble metal nanostructures, we have constructed the Au NPs30/V-Ti20/C.w. nanoarray to compare the Raman signal enhance performance and reproducibility with Ag15/V-Ti20/C.w. nanoarray. The TEM image of Au NPs with uniform size was shown in Fig. 4(a). By counting 58 random selected Au NPs in Fig. 4(a), the size of the Au NPs had a weak fluctuation with an average diameter of 30 nm as shown in Fig. 4(b). The FE-SEM image of Au NPs30/V-Ti20/C.w. substrate was exhibited in Fig. 4(c), large-scale Au NPs were decorated on the surface of the V-Ti20/C.w. nanoarray, which ensured the Au NPs30/V-Ti20/C.w. substrate gaining more appropriate sub-10 nm nanogaps with high-density electromagnetic enhancement “hot spots” for SERS performance. For the “hot spots”, the one using hot-carrier effects induced with MOS-like structure for field-induced emission enhancement can also achieve the Raman signal enhanced [39].
Fig. 4. (a) TEM image of Au NPs; (b) The size distribution of the Au NPs; (c) FE-SEM image of Au NPs30/V-Ti20/C.w. substrate.
3.4 SERS performance comparison of different substrates
It has been demonstrated that the type, size and morphology of noble metal NPs as well as the density of nanogaps had a crucial influence on the performance of SERS substrate [40]. In order to obtain the best SERS substrate which inspired the best LSPR performance, as well as determine the influence of the nanogap size on the SERS performance, R6G solutions with different concentrations were applied as probe molecule in SERS detection for its well established vibrational features [41].
Figure 5(a) exhibits the Raman spectra of 1 × 10−3 M R6G recorded from different types of substrates. The strong characteristic peaks of R6G were mainly centered at 611 cm−1, 774 cm−1, 1187 cm−1, 1361 cm−1, 1510 cm−1, 1572 cm−1 and 1648 cm−1 and the vibrational modes were given in Table 1 [42]. As shown in Fig. 5(b), different kinds of SERS substrates had significant differences in Raman signal enhancement when 10 µl R6G solution absorbed. We chose the typical characteristic peaks of 1187 cm−1, 1361 cm−1 and 1648 cm−1 to quantitatively describe the optimal SERS substrate. Obviously, the Ag15/V-Ti20/C.w. SERS substrate demonstrated won the best SERS performance. Table 2 shows the quantitative calculation of Raman signal enhancement degree of Ag15/V-Ti20/C.w. substrate and Au NPs30/V-Ti20/C.w. substrate at different characteristic peaks. From the quantitative calculation of the increase factors, we can conclude that the Raman signal enhancement performance of Ag15/V-Ti20/C.w. substrate was much higher than that of Au NPs30/V-Ti20/C.w. substrate. This positive result can be attributed to the regular nanorod-like structure on the surface of Ag15/V-Ti20/C.w. substrate and the regularly distribution of 10 nm nanogaps. Around the abundant 10 nm nanogaps, the LSPR effect was strongly generated and high-density electromagnetic enhancement “hot spots” were significantly increased. On the other hand, Au NPs on Au NPs30/V-Ti20/C.w. substrate had poor dispersion and could not form effective nanogaps that could stimulate LSPR effect. Therefore, the clustering of Au NPs further weakened the Raman signal intensity.
Fig. 5. (a) Raman spectra of 1 × 10−3 M R6G absorbed on different substrates; (b) Integrated Raman signal intensities of R6G at different characteristic peaks.
Table 1. Assignment of selected Raman peaks of R6G
Table 2. Calculation of increasing factors of different characteristic peak positions
3.5 Reproducibility of Ag15/V-Ti20/C.w. substrate
The high reproducibility of SERS substrate is the main challenge to be solved in its practical application. The reliability of SERS substrate ultimately depends on the uniform distribution of plasmonic noble metal materials on the SERS substrate [43]. The reproducibility of the prepared Ag15/V-Ti20/C.w. SERS substrate was verified by testing 25 random points from 5 Ag15/V-Ti20/C.w. substrate. The Raman spectra of R6G with a concentration of 1 × 10−4 M on Ag15/V-Ti20/C.w. substrate were shown in Fig. 6(a), and the corresponding characteristic peak intensities at 1510 cm−1 and 1648 cm−1 were exhibited in Fig. 6(b)–(c). The characteristic peak intensities of the peak at 1510 cm−1 fluctuated between 1489 and 1982 counts, and the characteristic peak intensities at 1648 cm−1 fluctuated between 5775 and 7810 counts. Although all the data showed fluctuations in Raman signal intensities, they still remained on the same order of magnitude. Next, we evaluated the reproducibility of SERS substrate by calculating the $RSD$ value on the basis of formula (1) [44]:
Fig. 6. Reproducibility test of 5 Ag15/V-Ti20/C.w. (a) and Au NPs30/V-Ti20/C.w. (d) SERS substrates; (b)-(c) Raman intensities of 1 × 10−4 M R6G at characteristic
The reproducibility test of the Au NPs30/V-Ti20/C.w. SERS substrate was also conducted by using 5 different Au NPs30/V-Ti20/C.w. substrates. The 25 measured Raman spectra of R6G which were random selected were shown in Fig. 6(d) and the corresponding distribution of Raman characteristic peak intensities at 1510 cm−1 and 1572 cm−1 were presented in Fig. 6(e)–(f). Obviously, the Raman characteristic peaks have great differences in intensity. The $RSD\; $ values for R6G at 1510 cm−1 and 1572 cm−1 were 10.81% and 10.94%. The reproducibility of Raman signal of Au NPs30/V-Ti20/C.w. substrate was worse than that of Ag15/V-Ti20/C.w. substrate. In the tests of reproducibility on these two different substrates, the RSD value < 7.31% of Ag15/V-Ti20/C.w. substrate can be attributed to the uniform distribution of Ag NPs over V-Ti20/C.w. nanorads, suitable nanogaps generated and good experimental operation of magnetron sputtering technology.
The above results fully demonstrate that the Ag15/V-Ti20/C.w. substrate attained the required high Raman signal enhancement and excellent reproducibility, and can be used for trace detection outside the laboratory.
3.6 Polarization property study of the Ag15/V-Ti20/C.w. substrate
Polarization effect has been proved to exist widely in the field of spectral analysis [1]. The influence of polarization dependence is almost inevitable in SERS applications, especially in anisotropic nanostructures. Sometimes, the polarization effect will have a negative impact on the SERS spectrum, such as reducing the reproducibility of the SERS signal [2]. Therefore, in the field of SERS detection, we should be aware of this phenomenon before interpreting the experimental results. In this work, the polarization dependence of Ag15/V-Ti20/C.w. substrate was firstly investigated. Figure 7(a) exhibited the principle diagram of polarization measurement of Ag15/V-Ti20/C.w. substrate. By constantly changing the direction of polarization, we recorded the Raman spectra at every 30° angle adjustment, and the corresponding Raman spectra were shown in Fig. 7(b). It can be seen that the Raman signal intensities hardly changed without red shift and blue shift. Figure 7(c)–(d) presented the Raman signal intensity at 1361 cm−1 and 1648 cm−1 characteristic peaks on the Ag15/V-Ti20/C.w. substrates with different polarization angles. The information in the Fig. 7(c)–(d) indicated that the Raman intensities of the major characteristic peaks showed no significant change when the polarization angle varied from 0° to 360° with 30° interval at a fixed macroscopic region. The Raman intensities at the characteristic peaks of 1361 cm−1 and 1648 cm−1 in polar coordinate showed a suborbicular polarized curve which further indicated that the Ag15/V-Ti20/C.w. nanoarray was polarization-independent.
Fig. 7. (a) The principle diagram of polarization measurement; (b) Raman spectra of R6G at different polarization angles on the Ag15/V-Ti20/C.w. substrate; (c)-(d) Polar curves of the Raman intensities at 1361 cm−1 and 1648 cm−1 characteristic peaks.
3.7 Sensitivity and AEF calculation of the Ag15/V-Ti20/C.w. substrate
Benefiting from a great quantity of uniform Ag NPs modified on the Ag15/V-Ti20/C.w. nanorods and the efficient LSPR response around the narrow 10 nm nanogaps, the Ag15/V-Ti20/C.w. substrate showed efficient trace detection capability for R6G with different concentrations. Figure 8(a) exhibits the Raman spectra of R6G with different concentrations varying from 1 × 10−5 M to 1 × 10−11 M recorded from the Ag15/V-Ti20/C.w. substrate. All the Raman characteristic peak intensities decreased with the decrease of R6G concentrations. When the concentrations of R6G solutions in Fig. 8(b1)–(b2) decreased to 1 × 10−10 M and 1 × 10−11 M, the characteristic peaks of 1361 cm−1 and 1648 cm−1 which assigned xanthenes ring stretching in plane C-H bending could still be identified, which indicated that the LOD for R6G on Ag15/V-Ti20/C.w. substrate was located on 1 × 10−11 M. The calibration plots for the Raman detection of R6G at 1361 cm−1 and 1648 cm−1 were shown in Fig. 8(c)–(d). In Fig. 8(c2)–(d2), the linear regression equations of different characteristic peaks were log I(1361 cm−1) = 0.2102(log C) + 5.74011 and log I(1648 cm−1) = 0.16875(log C) + 5.6566. The correlation coefficient (R2) values were of 0.98044 and 0.99213, respectively. The good linear regression equations indicated that the Ag15/V-Ti20/C.w. substrate has the ability to quantitatively detect other aromatic molecules and has great application prospects in the fields of biomedical sensing and drug pharmacodynamic substance detection.
Fig. 8. (a) Raman spectra of R6G with the concentration varies from 1 × 10−5 M to 1 × 10−11 M; Raman spectra of R6G in 1 × 10−10 M (b1) and 1 × 10−11 M (b2). (c1)-(d1) Raman signal intensities of R6G at different peaks; (c2)-(d2) Linear regression equations between the Raman signal intensity and R6G concentration.
To further evaluate the Raman signal enhancement performance of the Ag15/V-Ti20/C.w. substrate, the analytical enhancement factor ($AEF$) was calculated according to formula (2) [45,46]:
where ${I_{SERS}}$ and ${I_{Raman}}$ are the Raman signal intensities of R6G solution at the 1648 cm−1 characteristic peak on Ag15/V-Ti20/C.w. substrate and 1 × 10−2 M R6G solution on a silicon wafer, respectively, as shown in Fig. 9(a)–(b). ${C_{SERS}}$ and ${C_{Raman}}$ are the concentrations of R6G on Ag15/V-Ti20/C.w. substrate and on a silicon wafer, respectively. The experimental result in Fig. 8(a) showed that the minimum detectable concentration was 1 × 10−11 M. Therefore, this value was used in the calculation process of AEF. According to Eq. (2), the AEF value of the Ag15/V-Ti20/C.w. SERS substrate was 1.819 × 108. The ultra-high Raman signal enhancement ability of Ag15/V-Ti20/C.w. substrate can be comparable to the current international advanced EF value level, as shown in Table 3. Meanwhile, compared with the nanostructure SERS substrate we prepared previously [1,2,7,32,33,34], by optimizing the magnetron sputtering experimental parameters, the nanogaps between the Ag15/V-Ti20/C.w. nanorods were effectively regulated and the nanomorphology was more regular. The nanogap was within 10 nm, which was conducive to the excitation of strong Raman signal enhancement. Therefore, the Ag15/V-Ti20/C.w. substrate has a larger AEF value and smaller RSD values, thus, making the test result in practical application reliable.Fig. 9. (a) Raman spectra of 1 × 10−2 M R6G solution on the Si wafer; (b) Raman spectra of 1 × 10−11 M R6G solution on the Ag15/V-Ti20/C.w. SERS substrate.
Table 3. EF values on different SERS substrates with different nanostructures reported previously
3.8 Detection of Artemisinin by the Ag15/V-Ti20/C.w. substrate
Artemisinin is an important pharmacodynamic substance in Artemisia annua, and it has been called as “the only effective malaria treatment drug in the world at present” by the World Health Organization. With the continuous development of scientific research, Artemisinin have not only been proved to have unique efficacy in the treatment of malaria and schistosomiasis, but also proved to have anti-tumor, anti-inflammatory, immune regulation, antibacterial and other pharmacological effects [54–56]. Therefore, a fast and sensitive method for the determination of Artemisinin was essential. Based on the good Raman signal reproducibility, excellent sensitivity and remarkable Raman signal enhancement performance of Ag15/V-Ti20/C.w. substrate, in this paper, SERS technology was used for ultra-sensitive detection of Artemisinin content.
The variation of the Raman signal intensities of Artemisinin with diferent concentrations were shown in Fig. 10(a). The Raman signals observed at 823 cm−1, 1130 cm−1, 1258 cm−1, 1321 cm−1 and 1668 cm−1 were the fngerprint peaks of Artemisinin. Raman spectrum of 0.01 mg/l Artemisinin showed that the main peak at 1668 cm−1 could still be distinguished at this ultra-low concentration which indicated that the LOD of Artemisinin was estimated to be around 0.01 mg/l for the Ag15/V-Ti20/C.w. substrate. As shown in Fig. 10(b)–(c), when the concentration of Artemisinin and the intensity of Raman signal were simultaneously logarithmically calculated, a linear regression equation (log I(1668 cm−1) = 0.129751(log C) + 3.7061) was obtained with a R2 value of 0.97024. In a word, this high-performance SERS substrate can be used for rapid detection of other unlabeled pharmacodynamic substance in real traditional Chinese medicine.
Fig. 10. (a) Raman spectra of Artemisinin with different concentrations; (b) Raman signal intensities of Artemisinin at 1668 cm−1 peak; (c) Linear regression equation between the Raman signal intensity and Artemisinin concentration.
4. Conclusion
In this work, we present a novel SERS substrate based on Ag NPs modified V-Ti nanorads for the sensitive detection of R6G and Artemisinin. By strictly controlling the experimental parameters, we found the optimal SERS performance of Ag15/V-Ti20/C.w. substrate with tunble nanogaps. The Ag15/V-Ti20/C.w. SERS substrate displayed superior SERS performance for the detection of R6G, including the AEF of 1.819 × 108 and the LOD as low as 1 × 10−11 M. The nanoscale interparticle nanogaps of homogeneous Ag15/V-Ti20/C.w. nanorods can inspire strong LSPR effect under laser excitation to effectively strengthen the Raman signal by the electromagnetic mechanism. In the practical detection, the Raman spectra of Artemisinin and its linear regression equation were given with the LOD was located at 0.01 mg/l. javascript:void(0);The advantages of the constructed Ag15/V-Ti20/C.w. SERS platform in the current study proves its feasibility in the detection of pharmacodynamic substance in traditional Chinese medicine. This Ag15/V-Ti20/C.w. SERS substrate is promising to enable affordable quantitative analysis and provide a new strategy for the quality evaluation of pharmacodynamic substance of traditional Chinese medicine.
Funding
Technology and starting fund for scientific research of high-level talents of Chengde Medical University–Nature (202206); “Technology Innovation Guidance Project-Science and Technology Work Conference” of the Hebei Provincial Department of Science (none); Science and Technology Project of Hebei Education Department (QN2021236).
Disclosures
The authors declare no conflicts of interest.
Data availability
Data underlying the results presented in this paper are not publicly available at this time but may be obtained from the authors upon reasonable request.
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