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Abstract
Arrayed resonant cavity with outstanding optical trapping ability have received increasing attention in surface-enhanced Raman spectroscopy (SERS). Here, a three-dimensional (3D) composite AgNPs-Al2O3/Au/inverted patterned sapphire substrate PMMA (IPSSPMMA) flexible resonant cavity system is theoretically and experimentally investigated as a flexible SERS sensor. With the help of an effective plasma coupling (localized surface plasmons (LSPs) and surface plasmon polaritons (SPPs)), as shown by the Finite Element Method, a resonant cavity between IPSSPMMA and a particle-film nanostructure is created. Moreover, the proposed fabrication scheme can be easily used for large-scale fabrication. To measure the performance of IPSSPMMA, Rhodamine 6 G (R6G) and Crystalline violet (CV) were used as probe molecules with limit of detection (LOD) of 6.01 × 10−12 M and 5.36 × 10−10 M, respectively, and enhancement factors (EF) of R6G up to 8.6 × 109. Besides, in-situ detection of CV on the surface of aquatic products with a LOD of 3.96 × 10−5 M, enables highly sensitive in-situ detection of surface analytes. The Raman performance and in-situ detection results demonstrate that the proposed flexible compositing resonant cavity system has the advantages of ultra-sensitivity, stability, uniformity, and reproducibility, and has great potential for applications in the food safety field.
© 2023 Optica Publishing Group under the terms of the Optica Open Access Publishing Agreement
1. Introduction
Surface-enhanced Raman spectroscopy (SERS), a physical technology that can characterize molecular vibration, was vividly called the “fingerprint spectrum” of molecules by researchers, which set off a hot wave of scholars studying SERS [1,2]. In recent years, SERS has developed into a non-destructive, rapid, sensitive, specific detection method, and has emerged as a leader in biosensors [3–5], food safety [6–8], material science [9], medicine and catalysis fields [10,11]. The mechanisms of SERS enhancement include an electromagnetic mechanism (EM) and a chemical mechanism (CM) [12]. As for EM, it relies on the local field derived from the localized surface plasmon (LSP) resonance effect of noble metals (Au, Ag, and Cu). The local electric field will be further enhanced at the plasmonic “hot spot” region generated in the gaps of noble metal nanoparticles. In particular, it was demonstrated that the molecules located in the “hot spot” region accounted for only 0.01% of the total area, but contributed more than 25% of the SERS signal [13]. For CM, it is widely assumed that the charge-transfer (developing between the SERS substrate and the probe molecules) model plays a dominant role. Numerous investigations have shown that EM can provide 103-108 enhancement factors (EF), while CM is weaker and generally provides 102 enhancement factors [14–16]. It is especially important to design SERS structures that can produce high intensity and density “hot spot” regions because of the special benefits of EM in surface Raman enhancement.
Nanobowls [17], bowtie nanoantema [18] and nanopillar [18] have been designed for generating stronger LSP because of their richness in tips. Besides, some natural structures with evenly spaced gaps, such as mussel shells [19] and cicadan wings [20] are also widely used in SERS. As the design ideas of SERS substrates evolved from 2D to 3D, the concepts of multilayer particles and multilayer films were proposed [15]. By integrating LSP in the vertical direction, the multilayer particle system significantly increases the electric field enhancement effects. However, this system is difficult to prepare, performs inconsistently, and cannot be produced industrially, which restricts its growth. The surface plasmon polarizations (SPPs)-based film system, which is easier to prepare, exhibits better reproducibility and uniformity, but its enhancement effect is more widespread. The 3D composite substrate in multiple excitation modes is proposed to solve the above problems by combining the advantages of LSP and SPP. For example, Liu et al. designed an AuNPs-multilayer Au/Al2O3 hybrid structure as an efficient SERS substrate through the successful coupling of LSP and SPP and elucidated the relationship between the number of coupling material layers and the electric field enhancement effect by tuning the spacer layer thickness [21]. Besides, Kalachyova et al. designed novel 3D SERS structures by immobilizing various shapes of gold and silver nanoparticles (MeNPs) on Ag gratings, which can couple SPPs supported by silver gratings to LSPs excited on grafted MeNPs. This structure successfully achieved highly sensitive detection of 4,4′-biphenyldithiol (BFDT) at different wavelengths [22]. The AgNps-multilayer Al2O3/Au composite framework was developed by Zha et al. using the coupling of LSP and SPP to give the structure excellent SERS performance. And the low concentration detection of toluidine blue (TB) molecules was achieved, which effectively promotes the application of SERS substrate detection for staining biological cells [23]. However, the majority of these methods are challenging to scale up for industrial manufacturing.
The “hot spot” region of the SERS structure is sparsely populated, which reduces the likelihood that analyte molecules will get there. It seriously hinders the practical application of SERS. Research has concentrated on resonant cavity structures that offer extra degrees of freedom for “hot spots” through distorted spatial geometry SERS structures, considerably boosting the practical application of SERS. This work has been motivated by transformation optics (TO) [24–28]. Cavity structures can trap the laser, increase the light path, and further promote the interactions between photons and absorbed molecules [29]. For single molecule detection experiments, Mao et al. created a SERS structure with nanobowls that produced excellent Raman response and a quick immersion time [30]. Patterned Sapphire Substrate (PSS), in our search for a suitable substrate, slowly came into our awareness [31,32]. In the field of LEDs, PSS is a perfect solution to the “Droop” effect of ordinary sapphire substrate LEDs because of its customizable micron-level patterned microstructure, which can improve LED lifetime while scattering light from the active area multiple times, changing the angle of total reflection, increasing the light path of the incident light, and improving light extraction efficiency which is precisely the advantages needed for EM in SERS. The “lighting rod” effect, another geometric factor in addition to effective light utilization, also contributes to the enhancement of EM because the electromagnetic field of the nanostructure will be concentrated at the nanotips and edges [33]. In this case, quasi-3D SERS substrates with high curvature and conical or triangular structures can be used as active sites to create EM “hot spots” [34]. More importantly, PSS has been produced industrially with mature manufacturing technology and a low cost per piece. Compared with the Pyramid Si (PSI) substrate which is widely used in the SERS field now, PSS has a more uniform arrayed micromorphology and a smaller size, which undoubtedly increases the uniformity of the SERS substrate signal [35]. Thus, PSS is anticipated to replace PSI and be extensively utilized in SERS.
In this work, we successfully transferred the arrayed cone-like structure of PSS using PMMA, which had flexible and transparent properties and was promising for in-situ detection [36,37]. After the AgNPs-Al2O3/Au particles-film composite structure was deposited on PMMA, a 3D composite AgNPs-Al2O3/Au/inverted patterned sapphire PMMA (IPSSPMMA) flexible resonant system was successfully fabricated. Here, IPSSPMMA was used as a 3D flexible support layer instead of a rigid PSS. It was demonstrated by the Finite Element Method using COMSOL Multiphysics software that the 3D IPSSPMMA resonator cavity system improved the SERS performance more significantly than the PSS substrate did by contributing more to the local field enhancement. Raman experiments showed that the substrate could efficiently detect Rhodamine 6 G (R6G) and Crystal violet (CV) at low concentrations, and the limits of detection (LOD) was 6.01 × 10−12 M and 5.36 × 10−10 M for R6G and CV, respectively, with an EF of 8.6 × 109 for R6G. Additionally, the substrate showed an in-situ LOD of 3.96 × 10−5 M for CV molecules on aquatic products, illuminating the structure's exceptional capacity to find toxic materials during safety inspections.
2. Experimental section
2.1 Materials
Acetone (CH3COCH3, 99.5%) and alcohol (C2H6O, 99.7%) were purchased from the local chemical plant. Polymethyl meth acrylate (PMMA) was offered by the company. Au, Ag and Al targets materials were sourced from Fuzhou Invention photoelectrical Tech CO., Ltd. Rhodamine 6 G (R6G) and Crystal violet (CV) were purchased from Dalian Meilun Biological Technology Co., LTD and Shanghai Far Wild Science and Technology Co. LTD. The PSS substrates were purchased in Shanghai Ruipu Optical Materials Center (The microstructures are conical with a diameter of 2.4 µm, a height of 1.5 µm, and a gap between the microstructures of 0.5 µm.).
2.2 Fabrication of AgNPs-Al2O3/Au/IPSSPMMA SERS substrate
Figure 1 schematically and graphically illustrates the fabrication process of Ag NPs-Al2O3/Au/IPSSPMMA SERS substrate. First, 2 ml of PMMA/acetone (5 g/100 ml) was uniformly coated on the PSS substrate and heated for 40 min on a heating table at 40 °C. After heating, PMMA can be easily separated from the PSS substrate, and the obtained PMMA film has a valley cavity structure. Second, the obtained PMMA film is continuously deposited with uniform Au and Al layers by thermal evaporation (VZB-400). The Au film exhibiting a thickness of 50 nm was deposited on the PMMA film (deposition rate ∼0.2 Å/s). The Al film exhibiting a thickness of around 3 nm was deposited on the Au film (deposition rate ∼0.5 Å/s). The substrate rotated at a speed of 30 r/min during all deposition processes, which were conducted at a low pressure of 7 × 10−5 Pa. To obtain high-quality, dense aluminum film oxide, the samples were transferred to a high vacuum chamber protected by nitrogen and oxidized at low pressure (5 × 10−6 Torr) for 10 min. Dense alumina film can be produced on the Au film through repeated evaporation and oxidation, acting as a nanoscale gap and creating a hybrid structure with the Au film. After the hybrid structure was synthesized, the AgNPs were deposited on the dense Al2O3 film by thermal evaporation deposition. After a large number of experiments [21,23,38], we found that in the evaporation of 3.5 nm Ag film, if the current is 40 A and the deposition rate is 0.3 Å/s, the Ag film will become Ag NPs with a uniform size distribution.
2.3 Sample characterization
The structure and morphologies of the prepared samples were lucubrated by the scanning electron microscope (SEM) (ZEISS Sigma500 at 5.0 kV) with an energy-dispersive spectrometer (EDS) (at 20 kV operated at 200 kV).
2.4 SERS spectrum measurement
R6G molecules were successively diluted 10 times with alcohol solution to a concentration of 10−3-10−13, and so were CV molecules with a concentration of 10−3-10−11. To evenly expand the probe molecular solution on the surface of the medium without sliding down the substrate, 2 µL of the molecular solution was dropped on the surface of the substrate for natural drying before SERS detection was conducted. Raman spectrometers (Horiba HR Evolution) were applied to detect and capture the Raman signals. All of the Raman spectra were obtained using a 532 nm exciting laser, × 50 objective lens, 1µm laser spot, and 4 s of acquisition time. For the accuracy of the limit concentration detection, grating parameter of 1800 grooves per 1 mm was selected. The grating parameter used in other Raman spectral detection is 600 grooves per 1 mm, under which a large scanning range can be obtained, especially in the uniformity Mapping scanning, which will ensure the rigor and accuracy of the experiment.
2.5 Theoretical design and analysis
For an in-depth analysis of the SERS performance in the composite structure, the electric field distribution of the structure was simulated by the Finite Element Method. In this paper, the structural properties observed by electron microscopy are utilized to set the simulation parameters. As shown in Fig. 3 (a), the electric field distribution of only PSS and IPSSPMMA after inversion was first simulated with a conical height of 1.6 µm and a bottom radius of 1.2 µm for PSS and IPSSPMMA. Subsequently, inside the conical PMMA cavity, the structure of AgNPs-Al2O3/Au/IPSSPMMA was simulated with Ag NPs diameter of 30 nm, spacing of 10 nm, Au layer thickness of 50 nm, Al2O3 layer thickness of 6 nm, as shown in Fig. 3(b). The absorption boundary condition in the theoretical simulation is a perfectly matched layer. The sample is exposed to a 532 nm linearly polarized monochromatic plane wave that has been polarized along the x-axis. Physical field control mesh is the type of mesh, and the size is standard.
3 Results and discussion
3.1 Characterization of SERS substrate
The SEM pictures for the samples created by each of the aforementioned experimental stages are shown in Fig. 2. Compared with the purely typical PSS substrate in Fig. S1 (a), the arrayed structure shown in Fig. 2 (a) has been successfully transferred to the thin PMMA layer, forming an array of distributed inverted conical cavities constituting the resonant cavity structure. The fact that PMMA is colloidal during transfer makes this phenomenon simple to understand. When drop coated on a PSS substrate, PMMA will completely wrap around PSS and form a uniform film with a thickness of 20 µm. The PSS structure will be inverted in the PMMA film to create IPSSPMMA. After thermal vapor deposition of Au and Al2O3 films on IPSSPMMA, Fig. 2 (b) clearly shows the low-magnification surface view of the large-area arrayed pattern on IPSSPMMA, and the inset shows a magnified view of the details of the white circle area in the figure. This indicates that the Al2O3/Au/IPSSPMMA has been successfully and uniformly prepared. Additionally, we carried out high magnification SEM characterization to observe the AgNPs, as demonstrated in Fig. 2 (c) (high-magnification cross-sectional SEM image) and Fig. 2 (d) (high magnification surface SEM image). High magnification electron microscope observation allows for a clearer view of the cross section of the flexible AgNPs-/Al2O3/Au/IPSSPMMA substrate, as shown in Fig. 2 (c). In the figure, it can be seen that the nearly 50 nm Au film with a 6 nm Al2O3 film (brighter thin layer) has been successfully deposited into the IPSSPMMA inverted cone cavity, and not only that, it can also be seen that the AgNPs are uniformly deposited into the resonant cavity. In the figure, it is important to note that the irregularity of the cross section is due to the different ductility of the metal and PMMA when they are broken by liquid nitrogen treatment. As we can observe in the morphology and size distribution of the spherical particles of AgNPs in Fig. 2 (d), the AgNPs are uniformly distributed in the resonant cavity and the gap of the inverted cone, which will effectively enhance the local field. The AgNPs were uniformly distributed, and the histogram of diameter distribution was calculated based on SEM images. Their average diameter was close to 30 nm, while the spacing between AgNPs was typically 10 nm. Their diameters were primarily spread between 25 and 30 nm The uniform distribution of AgNPs ensure the uniform distribution and utilization efficiency of “hot spots”. Combining the EDS elemental maps of C, O, Al, Au, and Ag in Fig. 2 (e), we can identify that AgNPs-Al2O3/Au/IPSSPMMA substrates have been successfully prepared. The EDS mapping of the 3D flexible structure is shown in Fig. 2 (f), and the homogeneity of the Ag NPs, Al2O3, and Au films can be seen by observing the color distribution of the various components. By combining all these aspects of characterization, we have successfully prepared a 3D flexible compositing AgNPs-Al2O3/Au/IPSSPMMA resonator system. This resonant cavity structure increases the optical path of the incident light, increases the scattering cross-section of the scattered photons, and provides more efficient photon utilization than a planar SERS structure, so it will have higher SERS performance [39]. Additionally, the specific surface area is greatly increased by the 3D inverted cone shape, which also makes it easier for light to interact with the target molecule.
Fig. 2. SEM images: (a) SEM image of IPSSPMMA, (b) low-magnification well prepared arrayed Al2O3/Au/IPSSPMMA. Inset: High-magnification of the white-circle area in the figure. (c) Cross-sectional view (90°) of the flexible AgNPs-Al2O3/Au/IPSSPMMA substrate. (d) High-magnification SEM images of AgNPs-Al2O3/Au/IPSSPMMA and size distribution histograms of AgNPs. The EDS spectrum (e) and the EDS elemental maps (f) of the 3D flexible SERS substrate.
3.2 Simulation results and discussion
After inversion, the coupling effect of IPSSPMMA, AgNPs, Al2O3, and Au film significantly improves the SERS performance of the resulting substrates. We conducted a systematic investigation utilizing COMSOL Multiphysics software, motivated by the idea of fine enhancement. As shown in Fig. 3 (c) and Fig. 3 (d), although the EF of IPSSPMMA (∼1.42) and PSS (∼1.21) are approximately equal, the effective electric field enhancement area of IPSSPMMA is larger than that of PSS. The excellent capturing ability of IPSSPMMA for incident light will be more advantageous in the EM of SERS. Therefore, we decide to use IPSSPMMA as the substrate for our construction. As shown in Fig. 3(e), the electromagnetic field intensity on the AgNPs-Al2O3/Au/IPSSPMMA substrate is higher with a field enhancement factor of 28.1, which is roughly 20 times stronger than that of a single IPSSPMMA. Obviously, after adding the AgNPs-Al2O3/Au particle-film composite structure, the EM field is effectively localized in the resonant cavity, and numerous “hot spots” are generated at the gap, tip, and wall of the resonant cavity which will make the structure have excellent EM effect as shown in Fig. 3 (b1, b2, and b3). These findings suggest that the best field intensity enhancement and SERS performance can be achieved by combining a 3D IPSSPMMA resonant cavity structure with a particle-film composite structure. The resonant cavity structure formed by the inverted mold is widely used in SERS, and this study further confirms the excellent performance of the resonant cavity structure formed by the inverted mold structure in the field of SERS [30,40]. In this study, alumina served as a barrier between the AgNPs and Au film to create a hybrid structure, and the coupling of LSP and SPP produced a potent electric field [8,41]. A large enhancement of the local electromagnetic field is achieved by the strong coupling effect of the LSP generated between the AgNPs gap and the SPP on the Au film surface. Al2O3 is utilized in this instance as a spacer layer because of its strong light transmission and ability to reduce or even completely eradicate charge disorder in the surrounding dielectric medium. Numerous investigations have established that SPP is an interaction between electrons and electromagnetic waves on a metal surface. The collective oscillation of electrons at the metal/dielectric interface is a surface wave with energy propagating along the metal surface. Nevertheless, this energy decays exponentially in the direction perpendicular to the metal surface. Since it is influenced by the SPP field enhancement properties, it can be used as a physical enhancement mechanism for the preparation of SERS substrates. As a propagating, scattered electromagnetic wave, SPP has the potential to experience a momentum mismatch under specific circumstances. In addition, the laser in the medium or air cannot directly excite the SPP on the metal surface. For the above reasons, it is feasible to use the LSP generated by the incident laser excitation of AgNPs as a coupling mechanism to excite the SPP on the surface of gold thin films. The LSP can be excited by direct light irradiation because it is a non-propagating excited state in which the conduction electrons of the metal nanostructure are connected to the electromagnetic field. AgNPs can therefore operate as a nano-antenna-like structure to excite the SPP on the Au film surface by the LSP generated by the incident laser irradiation on AgNPs, which precisely resolves the issue of challenging SPP excitation [21]. As shown in Fig. 3 (f), the weak electric field in the figure without the addition of AgNPs indicates that no SPP is generated in the Al2O3/Au composite structure. The SPP in the underlying Au film is successfully excited by the LSP generated by the AgNPs in the resonant cavity of IPSSPMMA, and the LSP is strongly coupled with the SPP, resulting in a strong local field enhancement, as shown in Fig. 3 (b3), which shows the distribution of a strong electric field under the excitation of the incident laser in the Al2O3 spacer layer. The electric field is significantly enhanced as a result of the multiple excitation modes mentioned above coupling with one another, which supports the remarkably superior SERS performance of the flexible hybrid structure suggested in this study. Therefore, the optimum SERS performance can be perfectly achieved by the sophisticated design of 3D AgNPs-Al2O3/Au/IPSSPMMA flexible SERS substrates.
Fig. 3. Scheme for the COMSOL theoretical simulation in the PSS and IPSSPMMA (a) and AgNPs-Al2O3/Au/IPSSPMMA (b) samples. The electric field distribution for (c) PSS, (d) IPSSPMMA, (e) the composite SERS substrate, (f) Al2O3/Au/IPSSPMMA cavity wall, (b1) AgNPs/Al2O3/Au/IPSSPMMA gap, (b2) tip, and (b3) wall.
3.3 SERS performance of R6G and CV
The outstanding benefits of the proposed AgNPs-Al2O3/Au/IPSSPMMA flexible resonator system as a SERS sensor, such as ultra-sensitivity, uniformity, stability, large-scale manufacturing capability, and in-situ detection, are also investigated after theoretically demonstrating the optimal SERS performance. We selected R6G and CV as probe molecules to study the SERS capability of this substrate. Initially, the limits of detection (LOD) of R6G were inferred by studying the Raman spectra of different concentrations (10−13-10−7 M) of R6G molecules. The characteristic fingerprint peaks of 613, 773, 1188, 1363, 1506, and 1649 cm-1 of R6G can be observed from concentrations 10−13 M to 10−7 M in Fig. 4 (a). Besides, we can still easily identify the peak at 613 cm-1 at a concentration of 10−13 M, which depends on the vibrational coupling effect [42]. Figure 4 (b) presents a perfect linear relationship between signal intensity and R6G concentration at 613 cm-1. The linear equation is Log I = 0.332 Log C + 6.653, where I and C denote the Raman intensity and the concentration of R6G. The average signal intensity of the data was calculated using fifteen randomly chosen sampling points from the corresponding concentration sample, and the correlation coefficient (R2) value of 0.997 shows a high degree of credibility. Then the LOD of AgNPs-Al2O3/Au/IPSSPMMA flexible resonator system proposed in this study for R6G can be determined to be 6.01 × 10−12 M according to the formula: LOD = 3 (SD/S) [38].Besides the SERS performance of the substrate was assessed by calculating the enhancement factor (EF) : [41]
Where ISERS denotes the intensity of a 613 cm−1 peak of 10−13 M R6G on the composite SERS substrate, and IRaman is the intensity of 10−3 M R6G at the same wavelength on the blank PMMA as shown in Fig. 4 (c). NSERS and NRaman are the number of probe molecules gathered in the laser spot on the composite SERS substrate and the blank PMMA, respectively. To be specific, the spot diameters formed by 2 µL of R6G drops on the SERS substrate and blank PMMA were approximately 5 mm, and the signal intensities obtained from ISERS and IRaman were ≈209 and ≈243, respectively. As a result, the EF of this sample could be calculated as 8.6 × 109. Excellent uniformity and reproducibility are additional crucial factors to consider when determining whether the SERS sensor can be used in actual applications. Figure 4 (d) shows the SERS signals of 15 batches of AgNPs-Al2O3/Au/IPSSPMMA prepared under the same conditions as for R6G with the concentrations of 10−7 M. No significant difference was identified in the characteristic peak intensity of the R6G signal in the fifteen tests. As shown in Fig. 4 (e), the blue dot indicates the relative intensity of the peak at 613 cm-1 in the above spectrum. The red dashed line indicates the average degree with a relative standard deviation (RSD) value of 5.96%. This sample has excellent reproducibility, as seen in the figure. To evaluate the uniformity of the substrate, we collected Raman data of the 613 cm-1 peak in a 10 × 10 µm2 area on the substrate as shown in Fig. 4 (f). Following the aforementioned Raman spectroscopy tests for the R6G probe molecule detection characteristics, it is clear that the successfully constructed SERS active substrates, AgNPs-Al2O3/Au/IPSSPMMA, have excellent sensitivity, reproducibility, and repeatability for the detection of particular molecules. We also carried out the following tests to determine the specific detection capability of SERS substrates for CV molecules. At first, the LOD of CV was inferred by studying the Raman spectra of CV molecules at different concentrations (10−11-10−6 M). Moreover, in Fig. 5 (a), the characteristic fingerprint peaks at 422, 523, 728, 913, 1175, 1376, and 1620 cm-1 of CV were observed from concentrations 10−11 M to 10−6 M. The relative intensity of the 913 cm-1 peak was obtained from the above spectra and a linear function was constructed using the correlation coefficient (R2 = 0.990), as shown in Fig. 5 (b), which indicates an outstanding linear relationship between the Raman signal intensity and the CV concentration. This undoubtedly proves that: in this concentration range, the SERS substrate can be quantitatively detected for CV, and the variation of intensity is expressed by the empirical equation: Log I = 0.357 Log C + 6.382. In accordance with the method used to determine the LOD of R6G, the Raman signal intensity of various CV concentrations decreases with increasing concentration, and the LOD of CV can be calculated as 5.36 × 10−10 M.Fig. 4. (a) Raman spectra of R6G on the composite SERS substrate with different concentrations (10−7 to 10−13 M). (b) The interrelationship between the intensity at 613 cm−1 vs different R6G concentrations at log scale. (c) Raman spectrum of 10−11 M R6G on SERS substrate and that of 10−3 M R6G on blank PMMA substrate. (d) the SERS signals of 15 batches of AgNPs-Al2O3/Au/IPSSPMMA prepared under the same conditions for R6G with a concentrations of 10−7 M. (e) The average Raman intensity of R6G at 613 cm−1 from 15 different batches of the SERS substrates. (f) Raman mapping with an area of 10 × 10 µm2 for 613 cm−1 peaks.
Fig. 5. (a) Raman spectra of CV on the composite SERS substrate with different concentrations (10−11 to 10−6 M). (b) The interrelationship between the intensity at 913 cm−1 vs different CV concentrations at log scale. (c) the SERS signals of 15 batches of AgNPs-Al2O3/Au/IPSSPMMA prepared under the same conditions for CV with concentrations of 10−6 M.
Besides, we also examined the reproducibility of the SERS substrate for CV molecular detection. The SERS signals of 15 batches of AgNPs-Al2O3/Au/IPSSPMMA prepared under the same conditions at a concentration of 10−6 M in Fig. 5 (c) are shown. According to Fig. 5 (d), the intensities of the characteristic peaks of the above 15 CV spectra were compared, and the blue dots indicate the relative intensities of the peaks at 913 cm-1, and the red dashed line indicates the average intensity with a RSD of 5.59%. In this figure, it is clear that the Raman signal intensities of different batches of SERS samples for the same concentration of CV molecules are in perfect agreement, demonstrating the perfect reproducibility of the SERS substrate. Therefore, the composite resonant cavity of AgNPs-Al2O3/Au combined with IPSSPMMA has a high sensitivity to specific molecules. In addition, the simple and mass-produced preparation method enables excellent reproducibility of SERS substrates because IPSSPMMA has a perfect array arrangement which makes SERS substrates have excellent homogeneity. Thus, the composite resonant cavity SERS substrates combining AgNPs-Al2O3/Au and IPSSPMMA would be employed in the detection of toxic substances in practical applications.
3.4 Practical application: In-suit detection of biomolecule CV
IPSSPMMA is used as a flexible support layer in the AgNPs-Al2O3/Au/IPSSPMMA flexible resonant cavity system, which can be produced in large quantities in accordance with the template shape, as shown in Fig. 6 (a), which is a physical diagram of the fabricated SERS substrate (the shape of the template in the figure is circular, and the desired finished shape can be achieved by customizing the shape of the template or cutting the sample).
Fig. 6. (a) Photograph of AgNPs-Al2O3/Au/IPSSPMMA SERS substrate under natural light. (b) Photograph of the in-situ detection of CV on the surface of aquatic products. (c) SERS spectra of CV on the surface of aquatic products. (d) The interrelationship between the intensity at 913 cm−1 vs different CV concentrations at log scale in in-situ detection.
Combining the above SERS substrate characteristics, a promising AgNPs-Al2O3/Au and IPSSPMMA flexible resonant cavity system sensor can be applied for in-situ detection of biomolecule-containing solutions. CV is a typical triphenylmethane-type cationic dye used in practical applications and is a potent antifungal and antiparasitic in the aquaculture sector. The CV ion is recognized as a significant cation in the human body and is involved in a number of physiological processes. However, when these dyes enter the human body, their metabolites are highly residual and toxic in terms of carcinogenicity, teratogenicity and mutagenicity, which are extremely harmful to humans. As a result, higher CV ion concentrations in the human body have been linked to a number of illnesses, including cancer. Therefore, it is crucial to create a quick and accurate method for identifying CVs. To further investigate the application of composite SERS matrices in the field of in situ detection, we selected CV molecules to validate the biosensing performance of SERS matrices for in situ detection of CV content in aquatic products, as shown in Fig. 6 (b). The front of SERS substrate was fitted on the surface of aquatic products, which would facilitate CV molecules to enter the “hot spot” area, so as to realize the specific detection of CV molecules. When the 532 nm laser was shone directly onto the surface of clean aquatic products or the surface of aquatic products that had been soaked in 10−4 M CV solution for 12 hours, no obvious Raman signal was found, as shown in Fig. 6 (c). Moreover, when the flexible SERS substrate was glued to the surface of clean aquatic products, there was no characteristic peak of Raman signal of CV, which indicates that the CV content in the aquatic products we purchased is very small and will not be harmful to the human body. The aqueous products were coated with the flexible substrates after they had been submerged in 10−4, 10−5, and 10−6 M CV solutions for 12 hours. The distinctive Raman signature peak signals at 913 and 1620 cm-1 were observed. As shown in Fig. 6 (d), the relative intensity of the 913 cm-1 peak was obtained from in-suit detection and the linear function was constructed using the correlation coefficient (R2 = 0.977) which indicates an excellent linear relationship. This proves that: in this concentration range, the SERS substrate can be quantitatively detected for CV, and the variation of intensity is quantitatively expressed by the empirical equation: Log I = 0.357 Log C + 4.718. Under these circumstances, the LOD of in-situ detection for surface CV molecular of aquatic products was obtained at concentrations as low as 3.96 × 10−5 M. The AgNPs/Al2O3/Au particle-film composite layer can be easily reached by the laser thanks to the excellent transparency of PMMA, and the LSP and SPP are effectively coupled to form a lot of “hot spots” and amplify the Raman signal. In addition, after the laser passes through the particle-film composite layer, it will propagate repeatedly in the closed resonant cavity formed by the surface of the aqueous product and the SERS substrate, which increases the efficiency of laser utilization. Several findings clearly showed the practical value and potential applications of AgNPs-Al2O3/Au/IPSSPMMA substrates for the quick detection of surface analytes.
4. Conclusions
In conclusion, we have successfully fabricated AgNPs-Al2O3/Au/IPSSPMMA flexible resonator system SERS substrates using a simple, low-cost, and high-throughput method. The suggested SERS substrate has the ability to produce 3D dense “hot spots” that add to the reproductive, quantitative, and sensitive SERS signals. The electric field near the cavity wall is greatly enhanced by combining the excellent light-trapping capability of the IPSSPMMA resonant cavity with the effective coupling characteristics of the LSP and SPP in the AgNPs-Al2O3/Au particle-film system, as confirmed by simulations and experiments. A high SERS activity with the EF of 8.6 × 109 was achieved because of the excellent optical capture capability and abundant electromagnetic “hot spots”. According to the findings of several Raman experiments, the arrayed cavity distribution confers excellent uniformity and stability to the SERS substrate. These results indicate that our fabricated 3D AgNPs-Al2O3/Au/IPSSPMMA flexible SERS sensors can pave the way for SERS-based and in-situ detection applications with significant potential in the field of sensing and detection in biochemists.
Funding
National Natural Science Foundation of China (1167419, 12004226, 12074226); Natural Science Foundation of Shandong Province (ZR2018BF026).
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.
Supplemental document
See Supplement 1 for supporting content.
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