Fabrication of Multifunctional SERS Platform Based on Ag Nps Self

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Article Fabrication of Multifunctional SERS Platform Based on Ag NPs Self-Assembly Ag-AAO Nanoarray for Direct Determination of Pesticide Residues and Baicalein in Real Samples

Guochao Shi 1, Kuihua Li 1, Jungai Gu 1, Wenzhi Yuan 1, Shiqi Xu 1,*, Wei Han 2, Jianjun Gu 2, Liyong Wang 2,3,*, Zhibin Zhang 4, Congzhe Chen 1, Jialin Ge 1 and Mingli Wang 5,*

1 Department of Biomedical Engineering, Chengde Medical University, Chengde 067000, China; [email protected] (G.S.); [email protected] (K.L.); [email protected] (J.G.); [email protected] (W.Y.); [email protected] (C.C.); [email protected] (J.G.) 2 College of Physics and Electronic Engineering, Hebei Normal University for Nationalities, Chengde 067000, China; [email protected] (W.H.); [email protected] (J.G.) 3 School of Energy and Power Engineering, North University of China, Taiyuan 030051, China 4 College of Traditional Chinese Medicine, Chengde Medical University, Chengde 067000, China; [email protected] 5 State Key Laboratory of Materials Science & Technology and Key Laboratory for Microstructural Material Physics of Hebei Province, School of Science, Yanshan University, Qinhuangdao 066004, China * Correspondence: [email protected] (S.X.); [email protected] (L.W.); [email protected] (M.W.)

Abstract: Aiming at the shortcomings of high cost and time-consumption in traditional liquid chro- matography, an effective surface enhanced Raman scattering (SERS)-based trace detection method Citation: Shi, G.; Li, K.; Gu, J.; Yuan, has been proposed to quantitatively identify the active component of traditional Chinese medicine. W.; Xu, S.; Han, W.; Gu, J.; Wang, L.; In this paper, a high-performance and versatile SERS platform based on Ag nanoparticles (NPs) Zhang, Z.; Chen, C.; et al. Fabrication self-assembly Ag-anodized aluminium (Ag NPs-Ag-AAO) nanoarray was fabricated by control- of Multifunctional SERS Platform lable physico-chemical preparation technology. The results indicated that the electromagnetic ﬁeld Based on Ag NPs Self-Assembly enhancement effect was sharply strengthened as Ag NPs assembled, and the experimental enhance- Ag-AAO Nanoarray for Direct ment factor (EEF) value was calculated to be 1.0083 × 106. This novel Ag NPs-Ag-AAO nanoarray Determination of Pesticide Residues with substantial “hot spots” exhibited high SERS signal reproducibility, with the relative standard and Baicalein in Real Samples. Coatings 2021, 11, 1054. https:// deviation (RSD) value at less than 2.23%. More importantly, this SERS platform was applied to detect doi.org/10.3390/coatings11091054 active component Baicalein in Scutellaria baicalensis, and the limit of detection (LOD) was located at 10 fg/mL. Therefore, this Ag NPs-Ag-AAO nanoarray with high sensitivity, strong Raman signal Academic Editor: Angela De Bonis reproducibility and reliable practicability has broad application prospects in the rapid detection of trace substances in the active components of traditional Chinese medicine and is expected to Received: 3 August 2021 be popularized. Accepted: 25 August 2021 Published: 31 August 2021 Keywords: SERS; AAO; Ag nanopillar; Ag NPs; Baicalein; pesticide residue

Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affil- 1. Introduction iations. In visible and near-infrared light bands, noble metal (Cu, Ag, Au and Pt) composite nanoarrays can stimulate a strong surface plasmon resonance (SPR) effect. This effect improves the light scattering conversion efficiency of nonlinear optical processes that are sensitive to electromagnetic field intensity changes, of which the most representative one is Copyright: © 2021 by the authors. surface enhanced Raman scattering (SERS) technology [1,2]. Based on the advantages of Licensee MDPI, Basel, Switzerland. normal Raman detection, SERS technology effectively improves the intensity and sensitivity This article is an open access article of Raman signal, which can realize single molecule detection [3]. At the same time, it distributed under the terms and overcomes the problem that Raman signal is easily interfered by fluorescence signal; conditions of the Creative Commons therefore, it is widely used in surface science, food safety, public safety and biomedical Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ detection, among other fields [4–6]. 4.0/).

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Since the SERS phenomenon was discovered in 1974 [7], researchers have been com- mitted to revealing its enhancement mechanism. During this period, more than a dozen theoretical models have been put forward, and these numerous enhancement mechanisms can be mainly classified into two categories: electromagnetic enhancement (EM) mechanisms and chemical enhancement (CM) mechanisms [8]. It is generally believed that the EM mechanism contributed the most to the SERS enhanced effect. In recent years, the SPR model has been widely accepted to explain the EM mechanism [9]. According to the SPR model theory, when the incident light irradiates the rough surface of noble metal nanostructures, the free electrons will oscillate collectively at the interface between the noble metal and dielectric. If the frequency of incident light is the same as the natural frequency of free electron oscillation, the SPR effect will occur, which makes the electromagnetic field intensity of the noble metal surface significantly enhanced. When the probe molecule is in the electromagnetic field enhancement region, the Raman scattering cross section of the probe molecule will be significantly enhanced, and the obtained spectrum is the SERS spectrum of the probe molecule [10]. The CM mechanism suggests that the SERS phenomenon is caused by the change in the polarizability of the probe molecules adsorbed on the rough noble metal nanostructures. In CM mechanisms, the charge transfer model is widely accepted [11]. This charge transfer process will produce a resonance-like effect, which leads to an increase in the polarization of the probe molecule and the Raman scattering cross section, resulting in the enhancement of the Raman scattering signal intensity [12]. In most SERS systems, the mechanisms of EM and CM exist simultaneously and both contribute to the enhancement of the SERS effect. Theoretical and experimental studies have also confirmed that EM plays a major role in Raman signal enhancement (104–1012), while chemical enhancement usually contributes about 101–102 [13]. With the development of nanotechnology, researchers have developed a large number of noble metal nanostructures with different functions. The key to the vigorous development of SERS technology is the low cost and simple preparation technology. Because SERS technology can characterize the molecular information of the sample at the nanoscale and the highly sensitive and stable SERS active substrate can combine the molecular information with the plasmon characteristics of noble metal nanostructures, researchers prefer Au, Ag, Cu and other noble metal nanostructures, as these noble metals have large optical responses in the visible range [14–16]. Through reactive ion etching and ion sputtering technology, Prof. Liu prepared a kind of flexible and ordered Au nanocap-like SERS active substrate [17]. This flexible SERS substrate with periodic nanostructures has high sensitivity and reproducibility for the detection of organic molecules. Prof. Yang’s team prepared a kind of bioinspired Ag brochosomes-hollow microscale particle with submi- croscale pits-having broadband and omnidirectional SERS performance [18]. In 2019, this group assembled the double layer polyethylene microspheres colloidal crystal template by the self-assembly method and prepared a type of highly uniform volume enhanced Raman scattering (VERS) substrate by combining the thermal evaporation and reverse mode process [19]. Because the VERS substrate was composed of a microbowl and a hollow nanocone at the bottom of the bowl, the strong electromagnetic field was not only generated on the surface of the microbowl but also inside the hollow nanocone. Therefore, compared with the conventional SERS substrate, this periodic and regular geometry can achieve the highly reproducible and reliable detection of viruses and other macromolecular substances. In terms of SERS enhancement performance, Ag nanomaterials have a large optical response in the visible light range, a higher efficiency of enhanced Raman scattering and a simple geometric modulation structure, so they have been widely used in SERS detection, such as for graphene oxide film dip-coated Ag nanoarrays [20], Ag NPs-decorated Au nanorod arrays [21], Ag NPs-poly(acrylic acid-stat-acrylamide)-block-polystyrene nano-objects [22] and so on. At the same time, many research teams have developed active SERS substrates by using biomaterials as templates, such as Ag/Razor Clam [23], GO/Ag/Lotus leaf [24] and GO/Ag/cicada wing [25]. The surface of these biomaterials has a natural hierarchical structure, which not only provides abundant electromagnetic “hot spots” but also shows Coatings 2021, 11, x FOR PEER REVIEW 3 of 17

have developed active SERS substrates by using biomaterials as templates, such as Ag/Ra- zor Clam [23], GO/Ag/Lotus leaf [24] and GO/Ag/cicada wing [25]. The surface of these biomaterials has a natural hierarchical structure, which not only provides abundant electro- Coatings 2021, 11, 1054 3 of 17 magnetic “hot spots” but also shows super-hydrophobicity for the enrichment of analytes, thus obtaining strong electromagnetic field enhancement and high detection sensitivity. However, these noble metal nanostructured SERS substrates also have some disad- vantagessuper-hydrophobicity. For example, for the the contacts enrichment of noble of analytes, metal thus nanoparticles obtaining strong with electromagnetic the molecules and field enhancement and high detection sensitivity. environmental media will lead to an unnecessary charge transfer, which will affect the However, these noble metal nanostructured SERS substrates also have some disad- Ramanvantages. detection For example, results. the In contactsaddition, of the noble proportion metal nanoparticles of “hot spots” with thein the molecules total adsorption and surfaceenvironmental area of noble media metal will lead nanoparticles to an unnecessary is very charge small transfer, (<1%) [26] which, which will affect leads the to poor statisticalRaman detection results of results. ultra sensitive In addition, detection. the proportion Some ofSERS “hot substrates spots” in the also total have adsorption defects, such as surfacecomplex area manufacturing of noble metal process, nanoparticles high isequipment very small requirements, (<1%) [26], which high leads preparation to poor cost andstatistical low output, results which of ultra greatly sensitive limit detection. the application Some SERS of substrates regular noble also have metal defects, nanostructures such in asthe complex field of manufacturing SERS [27]. process, high equipment requirements, high preparation cost andAiming low output, at the which above greatly defects limit of the SERS application preparation of regular technology noble metal, innanostructures this work, we first in the field of SERS [27]. prepared an AAO template by anodizing high-purity aluminum foil after high-tempera- Aiming at the above defects of SERS preparation technology, in this work, we first tureprepared annealing an AAO and template chemical by electropolishing. anodizing high-purity Then, aluminum Ag nanopillar foil after arrays high-temperature were deposited onannealing the AAO and template chemical by electropolishing. magnetron sputtering Then, Ag to nanopillar construct arrays Ag-AAO were deposited nanostructures. on the The surfaceAAO templateof this nanoarray by magnetron was sputtering covered towith construct sub−10 Ag-AAO nm nanogaps, nanostructures. which The can surface excite uni- formof this and nanoarray high intensity was covered electromagnetic with sub−10 enhancement nm nanogaps, “ whichhot spots can excite”. Due uniform to the andexistence of highlarge intensity-scale nanocavities electromagnetic on the enhancement Ag-AAO nanoarray, “hot spots”. Ag Due NPs to thewith existence a diameter of large- of 30 nm werescale assembled nanocavities on onthe the Ag Ag-AAO-AAO (Ag nanoarray, NPs-Ag Ag-AAO) NPs nanoarray with a diameter by the of 30oil- nmwater were separa- tionassembled self-assembl on they Ag-AAOmethod, (Ag which NPs-Ag-AAO) can efficiently nanoarray improve by the the oil-water electromagne separationticself- field en- assembly method, which can efficiently improve the electromagnetic field enhancement hancement performance. The schematic diagram of the preparation process was shown in performance. The schematic diagram of the preparation process was shown in Figure1. FigureMore 1. importantly, More importantly, combined combined with the rapid with solvent the rapid pretreatment solvent pretreatment method, Ag NPs-Ag- method, Ag NPsAAO-Ag SERS-AAO substrate SERS substrate was used was for theused rapid for analysisthe rapid of analysis acephate of pesticide acephate residues pesticide on resi- duesthe on surface the surface of Scutellaria of Scutellaria baicalensis baicalensis, which improved, which theimproved efficient the and efficient time-saving and Raman time-saving Ramandetection detection method method of pesticide of pesticide residues. Meanwhile,residues. Meanwhile, the Ag NPs-Ag-AAO the Ag NPs SERS-Ag platform-AAO SERS platformcan quickly can andquickly accurately and accurately detect Baicalein detect under Baicalein neutral under pH conditionsneutral pH with cond a LODitions of with a LOD10 fg/mL, of 10 fg/mL, which is which expected is expected to open up to a newopen method up a new for the method identification for the of identification traditional of traditionalChinese medicine Chinese and medicine biomedical and sensing.biomedical sensing.

FigureFigure 1. Schematic 1. Schematic illustration illustration of of the the fabrication fabrication processprocess of of the the Ag Ag NPs-Ag-AAO NPs-Ag-AAO nanoarray nanoarray and the and SERS the measurement SERS measurement by by thethe Raman Raman system. system.

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2. Experimental 2.1.2. Experimental Chemicals and Materials 2.1. Chemicals and Materials The silver target (diameter: 60.0 mm, thickness: 2.0 mm, purity: 99.99%) and high purityThe aluminum silver target foil (diameter:(thickness: 60.0 0.25 mm, mm, thickness: purity: 99.999%) 2.0 mm, purity:were purchased 99.99%) and from high Nan- pu- changrity aluminum Hanchen foilNew (thickness: Materials 0.25 Technology mm, purity: Co., 99.999%) Ltd., Nanchang were purchased, China. The from 4- Nanchangaminothi- ophenol,Hanchen Methylene New Materials blue Technology(MB), oxalic Co., acid, Ltd., perchloric Nanchang, acid, China. acephate The, 4-aminothiophenol,crystal violet (CV) andMethylene anhydrous blue ethanol (MB), oxalic were all acid, analytical perchloric grade acid, and acephate, purchased crystal from violet J & K (CV)Scientific and anhy-Ltd., Beijing,drous ethanol China. wereThe N all-Hexane analytical was grade obtained and purchasedfrom the Damao from J Chemical & K Scientiﬁc Reagent Ltd., Factory Beijing, China. The N-Hexane was obtained from the Damao Chemical Reagent Factory in Tian- in Tianjin, China. Deionized water (18.25 MΩ) was used to prepare the solutions through- jin, China. Deionized water (18.25 MΩ) was used to prepare the solutions throughout out the experiment. the experiment.

2.2.2.2. Sample Sample Preparation Preparation 2.2.1.2.2.1. Pre Preparationparation of of Porous Porous AAO AAO Template Template FirstFirst of of all, all, we we assembled assembled a a thermostatic thermostatic electrolytic electrolytic cell cell to to prepare prepare a a porous porous AAO template.template. The The electrolyte electrolyte was was an an oxalic oxalic acid acid solution solution with with a a concentration concentration of of 0.3 0.3 M, M, and and thethe schematic schematic diagram diagram of of the the preparation preparation device device is shown is shown in Figure in Figure 2. First2., First,the high the purity high aluminumpurity aluminum foil was foil annealed was annealed in an annealing in an annealing furnace furnace at 400 at °C 400 for ◦ 2C h for and 2 h then and elec- then tropolishedelectropolished in the in mixture the mixture of perchloric of perchloric acid and acid anhydrous and anhydrous ethanol ethanol (volume (volume ratio 1:4) ratio for 51:4) min. for After 5 min. high After temperature high temperature annealing annealing and electro and- electro-chemicalchemical polishing, polishing, the high the purity high aluminumpurity aluminum foil was foil anodized. was anodized. Then, the Then, polished the polished aluminum aluminum foil was foilcleaned was with cleaned acetone with andacetone deionized and deionized water. After water. natural After drying, natural the drying, aluminum the aluminum foil was placed foil was in placed the thermo- in the staticthermostatic electrolytic electrolytic cell. The cell. oxidation The oxidation voltage voltage was between was between 40 V, the 40 V,oxidation the oxidation time timewas controlledwas controlled within within 300 s and 300 the s and constant the constant temperature temperature was 5 °C. was Finally, 5 ◦C. the Finally, AAO thenanofilm AAO cannanoﬁlm be obtained. can be obtained.

FigureFigure 2. 2. SchematicSchematic diagram diagram of of the the AAO AAO preparation preparation device. device.

2.2.2.2.2.2. Preparation Preparation of of Ag Ag-AAO-AAO Nanoarray Nanoarray TheThe Ag Ag-AAO-AAO nanoarray nanoarray was was prepar prepareded by high vacuum magnetron sputtering ( (RadioRadio frequency,frequency, Shenyang Shenyang Scientific Scientific Instruments Instruments Co., Co., Ltd Ltd.,., Shenyang, Shenyang, China China)) and an and ion an beam ion beamcom- positecomposite thin film thin deposition film deposition system system (FJL560, (FJL560, Shenyang Shenyang Scientific Scientific Instruments Instruments Co., Ltd Co.,., Shen- Ltd., Shenyang, China). First, the AAO template was fixed in the sputtering chamber. Before yang, China). First, the AAO template was fixed in the sputtering chamber. Before the sput- the sputtering, the plasma cleaning was carried out in the sputtering chamber with 5 sccm tering, the plasma cleaning was carried out in the sputtering chamber with 5 sccm argon that argon that flowed for 10 min. As the intensity of the pressure in the sputtering chamber flowed for 10 min. As the intensity of the pressure in the sputtering chamber reached 3.5 × 10−3 reached 3.5 × 10−3 Pa, the sputtering began. When the sputtering power was controlled at Pa, the sputtering began. When the sputtering power was controlled at 14 W and the MFC2 14 W and the MFC2 value in the flow indicator reached 50, the sputtering chamber built up value in the flow indicator reached 50, the sputtering chamber built up luminance success- luminance successfully. By controlling the sputtering time and sputtering power, we were fully. By controlling the sputtering time and sputtering power, we were able to obtain a series able to obtain a series of Ag-AAO nanoarrays with different morphologies. of Ag-AAO nanoarrays with different morphologies.

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2.2.3. Preparation of Ag NPs-Ag-AAO Nanoarray by Oil-Water Separation Self-Assembly Method First, we poured 1/3 volume of N-hexane into a clean petri dish. Then, the concen- trated Ag sol was slowly added to the N-hexane. The Ag sol (sphere) was evenly distributed in the upper layer of the N-hexane. In this paper, the classic Lee & Meise method [28] was used to prepare Ag sol. Then, the anhydrous ethanol was taken with a pipette and dropped on the mixture interface of the Ag sol and N-hexane. At this time, the Ag NPs would be adsorbed to the interface of the N-hexane. Under the induction of ethanol, the adsorbed Ag NPs would gradually increase and form a mirror-like Ag film, as shown in Figure1. The prepared Ag-AAO nanoarray was placed under the mirror-like Ag film with a tweezer and then lifted upward. The Ag film would remain on the Ag-AAO nanoarray, and a dense layer of Ag NPs formed on its surface. After natural drying, we used deionized water to clean the substrate three times to eliminate the influence of N-hexane and ethanol on the experimental results. Lastly, the composite structure of the Ag NPs-Ag-AAO nanoarray was constructed. What needs illustration is that the terminology to describe the samples of Ag-AAO fabricated at the sputtering time of x min was Agx-AAO substrate.

2.3. Characterization and SERS Measurements The morphology of the prepared AAO, Ag-AAO and Ag NPs-Ag-AAO were characterized by ﬁeld emission scanning electron microscopy (FE-SEM) (SU8220, Hitachi of Japan, Tokyo, Japan). The morphology and the size of the Ag NPs were characterized by the Trans- mission Electron Microscope (TEM) (HT7700, High-Technologies Corp., Ibaraki, Japan). UV-vis absorption spectra were obtained by the Shimadzu UV-2550 system (Shimadzu (China) Co., Ltd., Shanghai, China). The Raman spectra in this paper were all obtained by a Raman system (Horiba LabRAM HR800, HORIBA Jobin Yvon, Paris, France). During the SERS measurements, the laser power was 0.1 W, the laser wavelength was set at 532 nm, the objective lens of × 50 was used, the numerical aperture was 0.75, the acquisition time of each Raman spectrum was 10 s and the spectral resolution was 1 cm−1. Each probe molecule with a different concentration was prepared with deionized water by means of the dilution method with a factor of 10.

2.4. Electromagnetic Field Simulations Because the theory of Mie scattering is not applicable to the aggregation state of noble metal nanoparticles, nor is it applicable to the complex shape of nanostructures, the current commonly used method is the three-dimensional finite-difference time-domain (3D-FDTD) simulation method [29]. In this paper, the 3D-FDTD method was used to simulate the interaction between Ag nanostructures and electromagnetic fields, including the interaction between the irregular shape of Ag nanofilm and the size of Ag NPs with an excitation wave. The Ag nanomaterials studied in this paper belong to the dispersive materials, and the relative dielectric constant varies with frequency. According to the modified Drude model, the properties of the Ag nanomaterials were processed, and their relative dielectric constant can be expressed as:

εs − ε∞ σ ε(ω) = ε∞ + + = εr + iεi (1) 1 + iωτ iωε0 By expanding the Equation (1), the real and imaginary parts can be expressed as:

ε − ε ε = ε + s ∞ (2) r ∞ 1 + ω2τ2

(ε − ε )ωτ σ = s ∞ + εi 2 2 (3) 1 + ω τ ωε0

where the εs is the static relative permittivity, ε∞ represents the inﬁnite high frequency relative permittivity, τ is the conductivity and σ is the relaxation time. In order to ensure Coatings 2021, 11, x FOR PEER REVIEW 6 of 17

 s         (3) i 1  2 2  Coatings 2021, 11, 1054 0 6 of 17

where the εs is the static relative permittivity, ε∞ represents the infinite high frequency relative permittivity, τ is the conductivity and σ is the relaxation time. In order to ensure the reliability of the simulation results, in this paper, the parameters of Ag can refer to the reliability of the simulation results, in this paper, the parameters of Ag can refer to Gai’s theoreticalGai’s theoretical research research on the on modiﬁed the modified Drude Drude model model of wide of wavewide bandswave bands [30]. [30].

3. Results and Discussion 3.1. Morphology Characterization of the AAO Nanotemplate AAO has been widely used in the preparation of SERS substrates because of its highly uniform nanoporenanopore structure.structure. Once Once the the structure structure is combinedis combined with with the the Ag Ag nanomaterial, nanomaterial, the SPRthe SPR effect effect will will begenerated be generated on theon the edge edge ofthe of the nanopore, nanopore, thus thus ensuring ensuring the the formation formation of theof the high high density density “hot “hot spot”. spot”. On the On other the other hand, hand, the three-dimensional the three-dimensional ordered ordered nanopore na- structurenopore structure can also can effectively also effectively expand the expand speciﬁc the surface specific area surface of adsorbed area of analyteadsorbed molecules, analyte whichmolecules, is more which conducive is more toconducive the regulation to the of regulation electromagnetic of electromagnetic enhanced “hot enhanc spots”,ed “hot and thenspots”, optimize and then the optimize SERS performance the SERS performance of regular nanoarrays. of regular Figurenanoarrays.3a shows Figure the top-view3a shows FE-SEMthe top-view images FE- ofSEM the images as-prepared of the AAO as-prepared nanotemplate. AAO nanotemplate. The large-scale The nanoporous large-scale array na- wasnoporous formed array on the was surface formed of on the the AAO surface nanotemplate of the AAO with nanotemplate high uniformity with high and periodicity.uniformity Theand diameterperiodicity. of The the nanoporediameter of was the 50nanopore± 3 nm was and 50 the ± nanospace3 nm and the was nanospace about 140 was± about2 nm. Figure140 ± 23 nm.b exhibits Figure the 3b cross-sectionexhibits the cross view-section FE-SEM view image FE of-SEM AAO. image We canof AAO. observe We that can theob- single-channelserve that the single nanopores-channel on nanopores the surface on of AAOthe surface were highlyof AAO homogeneous, were highly homo withgeneous, a length ofwith 400 a ±length5 nm. of 400 ± 5 nm.

Figure 3. FEFE-SEM-SEM images of the prepared AAO nanoarrays from (aa)) toptop viewview andand ((bb)) cross-sectioncross-section view.view.

3.2. Nanomorphology of the Ag-AAOAg-AAO Nanoarrays and 3D-FDTD3D-FDTD Simulations According to the previous research [[31]31],, wewe concluded thatthat the magnetron sputtering technology has the advantages of a fast deposition rate, high purity, goodgood uniformityuniformity andand strong controllability. ByBy controllingcontrolling thethe keykey parametersparameters ofof sputteringsputtering timetime andand sputtering power, wewe regulatedregulated thethe enhancementenhancement effecteffect ofof the electromagnetic field,field, which depended on the distributiondistribution of “hot“hot spots”spots” inin thethe nanogaps.nanogaps. The FE-SEMFE-SEM imagesimages ofof thethe Ag-AAOAg-AAO nanoarrays withwith different different sputtering sputtering times times (and (and the the same same sputtering sputtering power) power) were displayedwere dis- inplayed Figure in4 Figurea–d. Because 4a–d. Because the AAO the prepared AAO prepared by anodization by anodization was highly was homogeneous, highly homogene- the Ag-AAOous, the Ag nanoarrays-AAO nanoarrays were also were highly also uniform highly after uniform sputtering after sputtering Ag nanofilms. Ag nanofilms. When the sputteringWhen the sputtering time was controlledtime was controlled at 10 min, at the 10 Agmin, nanofilms the Ag nanofilms began to began strictly to grow strictly at thegrow edges at the of edges the AAO of the nanopores. AAO nanopo Withres. theWith increase the increase in sputtering in sputtering time, time, the regularthe regular Ag nanofilmsAg nanofilms became became thicker thicker and thicker. and thicker. As shown As in shown Figure 4 inc, the Figure six-petal 4c, thenanoflower- six-petal ± likenanoflower nanostructure-like nanostructure appeared. Theappeared. average The length average of the length nanopetal of the nanopetal was 55 was3 nm 55 and ± 3 the average diameter of the nanopore was 40 ± 2 nm. Therefore, these rough six-petal flower-like nanostructures and nanopores could stimulate the potential SPR effect. More importantly, the average size of the nanogap between the nanopetals was less than 10 nm, resulting in a strong SPR coupling effect. Due to the regular distribution of these nanogaps, the Ag20-AAO (sputtering time was 20 min) nanoarrays can stimulate a uniform SPR effect and produce high-intensity electromagnetic “hot spots”. When the sputtering time Coatings 2021, 11, x FOR PEER REVIEW 7 of 17

nm and the average diameter of the nanopore was 40 ± 2 nm. Therefore, these rough six- petal flower-like nanostructures and nanopores could stimulate the potential SPR effect. Coatings 2021, 11, 1054 More importantly, the average size of the nanogap between the nanopetals was less7 ofthan 17 10 nm, resulting in a strong SPR coupling effect. Due to the regular distribution of these nanogaps, the Ag20-AAO (sputtering time was 20 min) nanoarrays can stimulate a uniform SPR effect and produce high-intensity electromagnetic “hot spots”. When the sput- wastering further time extendedwas further to 25exte min,nded as to shown 25 min, in Figureas shown4d, thein Figure regular 4d, six-petal the regular ﬂower-like six-petal nanostructureflower-like nanostructure disappeared, anddisappeared, the nanogaps, and whichthe nanogaps, were less which than10 were nm, less were than gradually 10 nm, ﬁlled by the sputtered Ag atoms. Therefore, the Ag -AAO (sputtering time was 20 min) were gradually filled by the sputtered Ag atoms.25 Therefore, the Ag25-AAO (sputtering nanoarraytime was 20 presented min) nanoarra irregulary presented island-like irregular nanostructures. island-like In thisnanostructures. case, the SPR In coupling this case, effectthe SPR was coupling reduced, effect which was led reduced, to the decrease which ofled SERS to the signal decrease strength. of SERS signal strength.

FigureFigure 4. 4. FE-SEMFE-SEM images of ( a)) Ag Ag1010-AAO,-AAO, (b (b) )Ag Ag1515-AAO,-AAO, (c) ( cAg) Ag20-20AAO-AAO and and (d) (dAg) Ag25-AAO25-AAO sub- substratesstrates with with different different sputtering sputtering times. times.

TheThe compositional compositional distribution distribution of of the the Ag Ag2020-AAO-AAO nanoarray nanoarray was was further further investigated investigated usingusing energy energy dispersive dispersive X-ray X-ray spectroscopy spectroscopy (EDX) (EDX) (Hitachi (Hitachi of Japan,of Japan, Tokyo, Tokyo, Japan). Japan Overlay). Over- mapslay maps of Ag of andAg and Al EDXAl EDX are are shown shown in Figurein Figure5a with5a with a clear a clea separationr separation and and uniform uniform distribution,distribution, which which confirmed confirmed the the Ag-coated Ag-coated AAO AAO nanostructure. nanostructure. The The cross-section cross-section view view FE-SEMFE-SEM imageimage ofof thethe Ag20--AAOAAO nanoarray nanoarray was was displayed displayed in in Figure Figure 5b.5b. We We can can see see that thatthe theas-sputtered as-sputtered Ag flower Ag flower-like-like nanostructures nanostructures consisted consisted of a nanopillar of a nanopillar roughly roughly 100 ± 6 100nm± in6 nmheight,in height, which which tended tended to be tovertically be vertically oriented oriented and anduniformly uniformly distributed distributed on onthe theAAO AAO surface. surface. AccordingAccording to to the the geometric geometric configuration configuration of of the the Ag Ag2020-AAO-AAO nanoarray nanoarray in in Figures Figures4c 4c andand5b, 5b, we we simulated simulated the the electromagnetic electromagnetic field field intensity intensity distribution distribution near near the the Ag Ag20-AAO20-AAO nanostructure.nanostructure. Figure Figure5c 5c was was the the Ag Ag2020-AAO-AAO structural structural model, model, in in which which the the top top diameter diameter of of thethe Ag Ag nanopillar nanopillar was was 55 55 nm nm and and the the height height of of the the nanopillar nanopillar was was 150 150 nm. nm. The The parameters parameters ofof the the simulationsimulation have been described described in in detail detail in in Section Section 2.4. 2.4 The. The continuous continuous sinusoid sinusoid laser laserwith witha wavelength a wavelength of 532 of nm 532 was nm chosen was chosen as the asincident the incident light in light our insimulation, our simulation, and the andlaser the perpendicularly laser perpendicularly propagated propagated into the model into the with model the polarization with the polarization direction of direction E. Figure of5d,eE. Figureshows5 thed,e electr showsomagnetic the electromagnetic field simulation field results simulation from resultsthe x-y fromplane the and x-y x-z plane plane. and x-z plane. There were dense nanogaps in the Ag20-AAO model, which formed multi- There were dense nanogaps in the Ag20-AAO model, which formed multi-level and uniform level and uniform “hot spots” with obvious local electromagnetic field enhancement. The “hot spots” with obvious local electromagnetic field enhancement. The simulation results of simulation results of the Ag20-AAO model showed that the maximum value of the local electromagnetic field was 42.91 V m−1. According to the following Equation (4) [32]:

4 Eloc(ω) GSERS = (4) Einc(ω) Coatings 2021, 11, x FOR PEER REVIEW 8 of 17

the Ag20-AAO model showed that the maximum value of the local electromagnetic field was 42.91 V m−1. According to the following Equation (4) [32]: 4 Coatings 2021, 11, 1054 8 of 17 Eloc  GSERS  (4) Einc 

where the EElocloc(ω)(ω) andand Eincinc(ω)(ω )areare the the EE andand EE00 inin the the 3D 3D-FDTD-FDTD calculations, calculations, respectively. respectively. × 6 Therefore, the theoretical enhancementenhancement factorfactor (TEF(TEF11) value was 3.39 × 10106. .

20 20 Figure 5. (a) EDX mapping of the Ag 20--AAOAAO nanoarray; nanoarray; ( (bb)) cross cross-section-section view FE FE-SEM-SEM image of the Ag20--AAOAAO nanoarray; nanoarray; (c) 3D-FDTD model of the Ag20-AAO nanoarray; the spatial distributions of the electromagnetic field intensity of the Ag20- (c) 3D-FDTD model of the Ag20-AAO nanoarray; the spatial distributions of the electromagnetic ﬁeld intensity of the AAO nanoarray in the (d) x-y plane and (e) y-z plane. Ag20-AAO nanoarray in the (d) x-y plane and (e) y-z plane.

In an effort toto improveimprove thethe electromagneticelectromagnetic fieldfield enhancementenhancement performanceperformance of thethe 20 20 Ag20--AAOAAO SERS SERS subs substrate,trate, the the Ag Ag NPs NPs were were assembled assembled onto onto the the Ag Ag-AAO20-AAO surface surface by the by oilthe-water oil-water separation separation self- self-assemblyassembly method. method. The TEM The TEMimage image of the of Ag the NPs Ag with NPs witha high a magnificationhigh magnification in Figure in Figure 6a revealed6a revealed that a that nanoparticle a nanoparticle count count obtained obtained from fromdifferent differ- re- gionsent regions of the ofsample the sample confirmed confirmed the presence the presence of essentially of essentially monodispersed monodispersed Ag NPs Ag adopt- NPs ingadopting a spherical a spherical-like-like morphology morphology with an with average an average diameter diameter of 30 nm. of 30 The nm. FE The-SEM FE-SEM image ofimage the Ag of theNPs Ag modified NPs modified Ag20-AAO Ag20 SERS-AAO substrate SERS substrate prepared prepared by the byoil- thewater oil-water separation sep- selfaration-assembl self-assemblyy method methodis shown is in shown Figure in 6b. Figure Ag 6NPsb. Ag with NPs a size with of a size30 nm of 30were nm closely were packedclosely packedon the surface on the of surface the Ag of20- theAAO Ag nanoarray20-AAO nanoarray and formed and a dense formed Ag a NPs dense nanolayer, Ag NPs asnanolayer, shown in as the shown Figure in the6b1 Figure. In order6b 1 .to In further order toinvestigate further investigate the electromagnetic the electromagnetic field en- hancementfield enhancement characteristics characteristics of the Ag of the NPs Ag30- NPsAg2030-AAO-Ag20 (30-AAO nm (30 Ag nm NPs Ag self NPs-assembl self-assemblyy Ag20- AgAAO20-AAO)) nanoarray, nanoarray, we constructed we constructed the theAg AgNPs NPs30-Ag30-Ag20-AAO20-AAO structure structure model, model, as shown as shown in Figurein Figure 6c.6 cThe. The intensity intensity distribution distribution of the of electromagnetic the electromagnetic field fieldnear nearthe Ag the NPs Ag30 NPs-Ag3020-- AgAAO20-AAO model model shown shown in Figure in Figure 6d,e indicates6d,e indicates that there that therewere werestrong strong electromagnetic electromagnetic fields infields the inwhole the whole space. space. Especially, Especially, high highelectromagnetic electromagnetic field fieldintensity intensity was wasconfined confined and anden- hancedenhanced at atthe the inter inter-Ag-Ag NPs NPs nanogaps. nanogaps. Meanwhile, Meanwhile, the the electromagnetic electromagnetic field field intensity at the Ag NPs-AgNPs-Ag nanofilmsnanofilms junctions can also be observed. According to the Equation (4), 9 the calculated TEFTEF22 was 2.8472.847 ×× 10109, ,which which was was 839.8 839.8 times times high than thatthat ofof thethe AgAg2020-AAO-AAO model. With thethe improvementimprovement of of the the nanomorphology nanomorphology of of the the Ag Ag20-AAO20-AAO nanoarray, nanoarray, the the Ag AgNPs NPs can can effectively effectively fill fill the the cavity cavity between between the the Ag Ag nanopillar, nanopillar, resulting resulting in thein the formation formation of ofabundant abundant sub sub−10−10 nm nm nanogaps. nanogaps. When When the the Ag Ag NPs NPs3030-Ag-Ag2020-AAO-AAO model was excited by the incident light, the effective Raman cross section increased and the collective resonance effect of the electrons enhanced. Therefore, the electromagnetic field intensity increased at the sub−10 nm gaps. Coatings 2021, 11, x FOR PEER REVIEW 9 of 17

Coatings 2021, 11, 1054 the incident light, the effective Raman cross section increased and the collective resonance9 of 17 effect of the electrons enhanced. Therefore, the electromagnetic field intensity increased at the sub−10 nm gaps.

1 30 20 FigureFigure 6.6. ((a)) TEMTEM imageimage ofof thethe AgAg NPs;NPs; ((bb,,bb1)) toptop viewview andand cross-sectioncross-section viewview FE-SEMFE-SEM images images of of the the Ag Ag NPs NPs30-Ag-Ag20-AAO-AAO substrate; (c) 3D-FDTD model of the Ag NPs30-Ag20-AAO nanoarray; the spatial distributions of the electromagnetic field substrate; (c) 3D-FDTD model of the Ag NPs30-Ag20-AAO nanoarray; the spatial distributions of the electromagnetic ﬁeld intensity of the Ag NPs30-Ag20-AAO nanoarray in the (d) x-y plane and (e) y-z plane. intensity of the Ag NPs30-Ag20-AAO nanoarray in the (d) x-y plane and (e) y-z plane.

3.3. SERS Performances of the Ag NPs30-Ag20-AAO Substrate 3.3. SERS Performances of the Ag NPs30-Ag20-AAO Substrate Referring to the wavelength position of the absorption peak in the UV-visUV-vis absorption spectrum, thethe optimaloptimal excitationexcitation wavelengthwavelength in RamanRaman measurement can be determined. Consequently, the the best best SERS SERS intensity intensity can can be obtained be obtained by choosing by choosing the best the excitation best excitation wave- 30 20 wavelengthlength [33,34] [33. Figure,34]. Figure 7a shows7a shows the UV the-vis UV-vis absorption absorption spectrum spectrum of the of Ag the NPs Ag NPs-Ag30-Ag-AAO20- AAOsubstrate. substrate. The absorp The absorptiontion peak center peak was center located was locatedat 524 nm, at 524 which nm, was which near was the near532 nm. the 532Therefore, nm. Therefore, in the SERS in the measurement, SERS measurement, we chose we the chose 532 the nm 532 laser nm as laser the as excitation the excitation light lightsource. source. TheThe SERS spectrum ofof thethe barebare AgNPsAgNPs3030-Ag-Ag20--AAOAAO substrate substrate has been detected first, ﬁrst, as shown in FigureFigure7 7b.b. TheThe characteristiccharacteristic peakpeak ofof thethe N-hexaneN-hexane and and ethanolethanol did did not not appearappear in thethe SERSSERS spectrum.spectrum. The N-hexaneN-hexane and ethanol werewere volatile.volatile. After assembling the AgAg NPs on the Ag 20--AAOAAO substrate, substrate, they they would would quickly quickly evaporate evaporate during during the the drying drying process. process. Therefore,Therefore, thethe inﬂuence influence of of N-hexane N-hexane and and ethanol ethanol on theon Ramanthe Raman spectroscopy spectroscopy were excluded.were ex- Figurecluded.7 cFigure shows 7 thec shows Raman the spectra Raman of spectra 10 −5 M of 4-aminothiophenol 10−5 M 4-aminothiophenol solution adsorbed solution ad- on differentsorbed on Ag-AAO different substrates Ag-AAO and substrates the Ag and NPs the30-Ag Ag20 NPs-AAO30-Ag substrate.20-AAO substrate. Meanwhile, Mean- the Ramanwhile, the spectrum Raman of spe thectrum 10−5 ofM the 4-aminothiophenol 10−5 M 4-aminothiophenol solution adsorbed solution on adsorbed the Ag NPs-AAOon the Ag substrateNPs-AAO was substrate also exhibited. was also Notably, exhibited. the Notably, Raman signal the Raman intensity signal of 4-aminothiophenol intensity of 4-amino- on Agthiophenol NPs30-Ag on20 Ag-AAO NPs was30-Ag stronger20-AAO thanwas thatstronger of the than 4-aminothiophenol that of the 4-aminothiophenol Raman intensities Ra- obtainedman intensities on the obtained Ag-AAO on nanoarrays the Ag-AAO and nanoarrays the Ag NPs-AAO and the substrate, Ag NPs-AAO owing substrate to the heavy, ow- self-assembleding to the heavy nature self-assembled of the Ag NPsnature by of the the oil-water Ag NPs separation by the oil- self-assemblywater separation method. self- Theassembl samey conclusionmethod. The was same also conclusion obtained in was the also calculation obtained of in the the Raman calculation characteristic of the Raman peak −1 integralcharacteristic area inpeak the integ rangeral of area 1502–1811 in the range cm , of which 1502– extracted1811 cm−1 from, which the extracted Raman spectra from the in FigureRaman7 c,spectra as shown in Figure in Figure 7c, as7d shown1–d4. Inin orderFigure to 7 quantitativelyd1–d4. In order analyze to quantitatively the enhancement analyze performancethe enhancement of the performance Ag NPs30-Ag of20 the-AAO Ag SERSNPs30 substrate,-Ag20-AAO the SERS increase substrate, factor valuesthe increase were calculated in Table1. From the calculation results of the increase factors, it can be concluded that the SERS enhancement effect of the Ag NPs30-Ag20-AAO substrate was much higher than that of the Agx-AAO substrates. This result can be attributed to the wide range of “hot spots” density introduced by the Ag NPs after self-assembly. Compared with the more Coatings 2021, 11, x FOR PEER REVIEW 10 of 17

factor values were calculated in Table 1. From the calculation results of the increase fac- Coatings 2021, 11, 1054 tors, it can be concluded that the SERS enhancement effect of the Ag NPs30-Ag2010-AAO of 17 substrate was much higher than that of the Agx-AAO substrates. This result can be attributed to the wide range of “hot spots” density introduced by the Ag NPs after self- assembly. Compared with the more dispersed Ag NPs, as shown in Figure 6a, the Ag NPs dispersed Ag NPs, as shown in Figure6a, the Ag NPs on the Ag NPs 30-Ag20-AAO SERS substrateon the Ag preparedNPs30-Ag20 by-AAO the self-assemblySERS substrate method prepared showed by the slightself-assembly aggregation, method which showed can furtherslight aggregation, facilitate the which formation can further of more facilitate “hot spots” the toformation enhance theof more SERS “hot response. spots” to enhance the SERS response.

FigureFigure 7.7. ((aa)) UV-visUV-vis extinctionextinction spectraspectra of of the the Ag Ag NPs NPs3030-Ag-Ag2020-AAO-AAO substrate;substrate; ((bb)) RamanRaman spectraspectra ofof thethe bare bare AgNPs AgNPs3030-Ag-Ag2020- AAOAAO substrate;substrate; ((cc)) RamanRaman spectraspectra ofof thethe 4-aminothiophenol4-aminothiophenol onon differentdifferent types types of of SERS SERS substrates; substrates; ( d(d1–1–dd44)) thethe integratedintegrated −1 −1 integralintegral areaarea ofof thethe RamanRaman shiftshift inin thethe rangerange ofof 15021502 cmcm−1––18111811 cm cm−.1 .

Table 1. Raman intensities of differentTable SERS 1. Raman substrates intensities at the of peak different of 1582 SERS cm− substrates1. at the peak of 1582 cm−1. Peak Intensity I (cps) Increase Factor Sputtering Time (min)Peak Intensity I (cps) Increase Factor Sputtering Time (min) IAgx-AAO IAg NPs30-Ag20-AAO (IAg NPs30-Ag20-AAO/IAgx-AAO) I I (IAg NPs30-Ag20-AAO/IAgx-AAO) 10Agx-AAO 182,101.97 ± 5480.78Ag NPs30-Ag20-AAO 1.037 × 106 ± 159.3 5.69 ± 0.029 10 182,101.9715 ± 5480.78 296,142.98 ± 3581.301.037 × 106 1.037± 159.3 × 106 ± 159.3 5.693.50± 0.029 ± 0.044 15 296,142.9820 ± 3581.30 407,642.04 ± 6139.091.037 × 106 1.037± 159.3 × 106 ± 159.3 3.502.54± 0.044 ± 0.026 20 407,642.04 ± 6139.09 1.037 × 106 ± 159.3 2.54 ± 0.026

3.4. Sensitivity of the Ag NPs30-Ag20-AAO Substrate

3.4. SensitivityAs a complete of the Agstudy NPs on30-Ag the20 preparation-AAO Substrate and application of the SERS substrate, the potentialAs a application complete study in sensitivity on the preparation detection of and the application as-proposed of SERS the SERS-based substrate, platform thefor potentialquantitatively application analyzing in sensitivity the MB molecule detection was of investigated the as-proposed by directly SERS-based adsorbing platform the MB for quantitativelymolecule on the analyzing Ag NPs30 the-Ag MB20-AAO molecule substrate. was investigated Figure 8a exhibits by directly the sensitivity adsorbing response the MB −7 moleculeof the Ag onNPs the30-Ag 20 NPs-AAO30-Ag substrate20-AAO to substrate. the MB molecule Figure8a with exhibits the conc the sensitivityentrations responsefrom 10 −12 ofM theto 10 Ag NPsM. The30-Ag Raman20-AAO spectra substrate detection to the results MB molecule showed withthat the Raman concentrations characteristic from 10peaks−7 M located to 10− 12at M.768 The cm Raman−1, 1155 spectracm−1, 1396 detection cm−1 and results 1626 showedcm−1 can that be observed the Raman in charac-Figure teristic8a, and peaksthe corresponding located at 768 vibration cm−1, 1155 modes cm− [35]1, 1396 of these cm−1 Ramaand 1626n characteristic cm−1 can be peaks observed were incollected Figure 8ina, Table and the 2. The corresponding characteristic vibration peak intensity modes [at35 1626] of thesecm−1 was Raman used characteristic to indirectly peaksmonitor were the collectedcorresponding in Table concentrations2. The characteristic of the MB peak molecule. intensity As at expected, 1626 cm −the1 was SERS used in- totensity indirectly of the monitor Ag NPs the self corresponding-assembly SERS concentrations substrate increased of the MBwith molecule. the increase As of expected, the MB themolecular SERS intensity concentration of the Agin the NPs range self-assembly of 1 × 10−12 SERS M−1 substrate × 10−7 M. increased At the same with time, the increase a good oflinear the MBrelationship molecular was concentration established inbetween the range the oflogarithm 1 × 10− 12ofM SERS−1 × intensities10−7 M. Atat 1626 the same cm−1 time,and the a good logarithm linear relationshipof MB concentrations was established, as shown between in Figure the logarithm 8b. The of linear SERS calibration intensities − atcurve 1626 was cm fitted1 and as the y = logarithm 7.104 + 0.258, of MB x (R concentrations,2 = 0.98636). Error as shownbars in inthe Figure plot represented8b. The linear the calibration curve was ﬁtted as y = 7.104 + 0.258, x (R2 = 0.98636). Error bars in the plot represented the standard deviations from ﬁve measurements of different spots for each concentration. Therefore, the LOD of the MB molecule on the Ag NPs -Ag -AAO was 30 20 1 × 10−11 M. Here, the effect of the pre-resonance Raman enhancement performance on Coatings 2021, 11, x FOR PEER REVIEW 11 of 17

Coatings 2021, 11, 1054 11 of 17 standard deviations from five measurements of different spots for each concentration. Therefore, the LOD of the MB molecule on the Ag NPs30-Ag20-AAO was 1 × 10−11 M. Here, the effect of the pre-resonance Raman enhancement performance on LOD was not consid- LOD was not considered. These results indicated that the proposed SERS-based platform ered. These results indicated that the proposed SERS-based platform had good potential had good potential for the practical detection in real samples of other aromatic molecules, for the practical detection in real samples of other aromatic molecules, even pesticide res- even pesticide residues and active ingredients of Traditional Chinese medicine. idues and active ingredients of Traditional Chinese medicine.

Figure 8. (a) Raman spectra of the MB solution with different concentrations on the Ag NPs30-Ag20-AAO substrate; (b) the Figure 8. (a) Raman spectra of the MB solution with different concentrations on the Ag NPs30-Ag20-AAO substrate; −1 (linearb) the relationship linear relationship between between the integral the integral area of Raman area of intensity Raman intensity and the different and the different concentrations concentrations at 1626 cm at 1626 in a cmlogarithm−1 in a scale. logarithm scale.

Table 2. Vibrational modes of the MB Raman characteristic peaks. Table 2. Vibrational modes of the MB Raman characteristic peaks. Raman (cm−1) SERS (cm−1) Vibrational Mode Raman (cm−1) SERS (cm−1) Vibrational Mode 596 (w) 595 (w) Skeletal deformation of C–S–C 671596 (w) (w) 672 (w) 595 (w)Out Skeletal-of-plane deformation bending of of C C–S–C–H 770671 (w) (w) 768 (w) 672 (w) Out-of-planeIn-plane bending bending of C– ofHC–H 770 (w) 768 (w) In-plane bending of C–H 1154 (w) 1155 (w) In-plane bending of C–H 1154 (w) 1155 (w) In-plane bending of C–H 1394 (m) 1396 (m) Symmetrical stretching of C–N 1394 (m) 1396 (m) Symmetrical stretching of C–N - -1468 1468(w) (w)Asymmetrical Asymmetrical stretching stretching of ofC– C–NN 16231623 (s) (s)1626 1626(s) (s)Ring Ring stretching stretching of ofC– C–CC NotationNotation:: s, s strong;, strong; m, m medium;, medium; w, weak w, weak peak intensity.peak intensity.

30 20 3.5. R Reproducibilityeproducibility of the Ag NPs 30--AgAg 20-AAO-AAO Substrate Substrate In practical application, another important content of Raman scattering research is the reproducibility of the SERS signals. In many cases, random distribution or a lack of electromagnetic “hot spots” can lead to a great deviation in the SERS signal signal streng strengthth [36] [36].. Therefore, the the reproducibility reproducibility of of the the Raman Raman signal signal greatly greatly affected affected the reliability the reliability and prac- and practicabilityticability of the of SERS the SERS system system detection. detection. In order In orderto further to further reveal revealthe Raman the Raman signal signalrepro- ducibilityreproducibility and practicability and practicability of the Ag of the NPs Ag30-Ag NPs20-30AAO-Ag20 substrate,-AAO substrate, the Raman the detection Raman de- of acephatetection of on acephate the surface on theof Scutellaria surface of baicalensisScutellaria was baicalensis carried wasout by carried using out the by“pasted using and the “pastedpeeled off” and method. peeled off” First, method. Scutellaria First, baicalensisScutellaria was baicalensis thoroughlywas thoroughly washed with washed deionized with waterdeionized and anhydrous water and anhydrousethanol. Then, ethanol. 10 μL Then, of 10−10 10 mg/mLµL of 10 acephate−10 mg/mL solution acephate were solutionsprayed weredirectly sprayed on the directly surface onof Scutellaria the surface baicalensis of Scutellaria. After baicalensis natural. evaporation After natural at evaporation room temper- at ature,room temperature,the 10 μL anhydrous the 10 µL ethanol anhydrous solution ethanol was solution dropped was onto dropped the pre onto-treated the pre-treatedsample. In thissample. step, In the this ethanol step, the act ethanoled as an acted extraction. as an extraction.Finally, the Finally, Ag NPs the30- Ag20 NPs-AAO30-Ag SERS20-AAO sub- strateSERS substratewas pressed was onto pressed the sample onto the for sample 30 s and for then 30 s peeled and then off peeledfor further off forRaman further detec- Ra- tion.man This detection. process This was process repeated was five repeated times to ﬁve ensure times the to successful ensure the collection successful of collectionacephate. of acephate.Acephate is a kind of organophosphate pesticide. In agricultural production, acephateAcephate is often is aused kind as of a organophosphate protective fungicide pesticide. for leafy In agricultural vegetables, production, traditional acephateChinese medicine,is often used tea asand a protectivecorn. This fungicidepesticide can for leafyinhibit vegetables, the activity traditional of cholinesterase Chinese in medicine, the hu- mantea and body, corn. resu Thislting pesticide in neurophysiological can inhibit the dysfunction. activity of cholinesterase Once inhaled, in acephate the human can cause body, resulting in neurophysiological dysfunction. Once inhaled, acephate can cause poisoning and even death. Therefore, the Ag NPs30-Ag20-AAO SERS substrate was applied to detect

Coatings 2021, 11, x FOR PEER REVIEW 12 of 17

poisoning and even death. Therefore, the Ag NPs30-Ag20-AAO SERS substrate was applied to detect the acephate residue on Scutellaria baicalensis. Figure 9a shows the substrate-to- substrate reproducible SERS spectra of 10−10 mg/mL acephate. These 25 SERS spectra were obtained from 25 points randomly selected from the 5 Ag NPs30-Ag20-AAO substrates. From the Raman spectra in Figure 9a, we found that the characteristic peaks of acephate did not shift to red or blue, and the signal intensity of the characteristic peak showed no difference. The relative standard deviation (RSD) values were calculated to evaluate the reproducibility on the basis of Equation (5) [37]: Coatings 2021, 11, 1054 12 of 17 n 2  IIi   i1 (5) RSD  n 1 the acephate residue on Scutellaria baicalensis. FigureI 9a shows the substrate-to-substrate reproducible SERS spectra of 10−10 mg/mL acephate. These 25 SERS spectra were obtained wherefrom Ī 25 is points the average randomly intensity selected of from all of the the 5 AgSERS NPs spectra30-Ag20,-AAO n is 25 substrates. and Ii is the From intensity the of eachRaman SERS spectra spectrum in Figure on 9thea, we same found characteristic that the characteristic peak. The peaks RSD of values acephate at did the not characteristic shift peaksto red of or 1356 blue, cm and−1 theand signal 1572 intensitycm−1 were of theexhibited characteristic in Figure peak 9b,c. showed Each no difference.RSD value The was less thanrelative 2.23%, standard which deviation fully revealed (RSD) valuesthat the were Ag calculated NPs30-Ag to20- evaluateAAO substrate the reproducibility had a good on repro- the basis of Equation (5) [37]: ducibility of the Raman signal in the wholes region. In order to further confirm the point- n 2 to-point reproducibility of the Ag NPs30-Ag20∑-(AAOIi−I) substrate, a randomly selected 5 μm × i=1 2 n−1 5 μm = 25 μm area was chosen, andRSD the= laser scanning step was 1 μm. The point-(5)to-point Raman mapping image at 1181 cm−1 is shown Iin Figure 9d. The brightness of the grid was −1 proportionalwhere I¯ is the to average the Raman intensity signal of intensity all of the SERSat 1181 spectra, cm , nwhichis 25 andindicatedIi is the that intensity the Ag of NPs30- Ageach20-AAO SERS substrate spectrum had on the an same excellent characteristic Raman peak.signal The reproducibility.RSD values at the characteristic −1 −1 peaksDue of to 1356 the cm excellentand 1572reproducibility cm were exhibited and reliable in Figure practicability9b,c. Each ofRSD the valueAg NPs was30-Ag20- AAOless thansubstrate, 2.23%, the which Raman fully spectra revealed of that acephate the Ag were NPs30 successfull-Ag20-AAOy substrate detected had at an a good extremely reproducibility of the Raman signal in the whole region. In order to further conﬁrm the low concentration level (10−10 mg/mL). Compared with the limit of quantitation (LOQ) point-to-point reproducibility of the Ag NPs30-Ag20-AAO substrate, a randomly selected specified5 µm × in5 µthem =national 25 µm2 foodarea wassafety chosen, standard and of the China laser scanning(GB 2763 step-2014), was the 1 µdetectionm. The limit ofpoint-to-point the Ag NPs30 Raman-Ag20- mappingAAO SERS image substrate at 1181 cmwas−1 isfar shown lowe inr than Figure the9d. LOQ. The brightness It was further provedof the gridthat wasthe proportionalAg NPs30-Ag to20 the-AAO Raman substrate signal intensityhas super at sensitivity 1181 cm−1, whichand wins indicated a wide ap- plicationthat the prospect Ag NPs30 -Agin actual20-AAO industrial substrate hadand anagricultural excellent Raman production. signal reproducibility.

FigureFigure 9. (a 9.) SERS(a) SERS spectra spectra of of 10 10−10− 10mg/mLmg/mL acephate acephate obtained obtained from 2525 randomly randomly selected selected spots; spots; (b,c )( Ramanb,c) Raman characteristic characteristic peakpeak intensities intensities of 10 of−10 10 −mg/mL10 mg/mL acephate; acephate; (d) (d SERS) SERS mapping mapping image image ofof thethe 1181 1181 cm cm−1−1characteristic characteristic peak. peak.

Due to the excellent reproducibility and reliable practicability of the Ag NPs30-Ag20- AAO substrate, the Raman spectra of acephate were successfully detected at an extremely low concentration level (10−10 mg/mL). Compared with the limit of quantitation (LOQ) speciﬁed in the national food safety standard of China (GB 2763-2014), the detection limit of the Ag NPs30-Ag20-AAO SERS substrate was far lower than the LOQ. It was further proved Coatings 2021, 11, 1054 13 of 17

that the Ag NPs30-Ag20-AAO substrate has super sensitivity and wins a wide application prospect in actual industrial and agricultural production.

3.6. Experimental Enhancement Factor (EEF) Calculation In the above description, we used the 3D-FDTD method to simulate the electromag- 9 netic ﬁeld enhancement near the Ag NPs30-Ag20-AAO model, and the TEF was 2.847 × 10 . In order to further evaluate the Raman enhancement performance of the Ag NPs30-Ag20- AAO SERS substrate from an experimental point of view, the experimental enhancement factor (EEF) value was calculated according to the following Equation (6) [38]:

I N EEF = SERS × bulk (6) Ibulk NSERS

−2 where the Ibulk and ISERS are the Raman signal intensities of 10 M CV solution on the Si −6 −1 wafer and 10 M CV solution on the Ag NPs30-Ag20-AAO SERS substrate at the 1619 cm characteristic peak. Nbulk and NSERS represent the number of CV molecules on the Si wafer and the Ag NPs30-Ag20-AAO SERS substrate under the laser spot. Figure 10a,b exhibits the Raman spectra of 10−2 M CV solution on the Si wafer and 10−6 M CV solution on the Ag NPs30-Ag20-AAO SERS substrate, respectively. Through the integral calculation of the −1 1619 cm characteristic peak area, the values of Ibulk and ISERS were 26,314 and 3,184,950. Therefore, the value of ISERS/Ibulk was 121.036. The number of CV molecules (N: Nbulk and NSERS) can be calculated according to Equation (7) [39]: NA × M × Vsolution N = × Slaser (7) Ssub

where NA is the Avogadro constant, M is the CV molecular concentration and Vsolution = 10 µL is the volume of the CV solution on the Si wafer and the Ag NPs30-Ag20-AAO SERS substrate. Ssub stands for the area of the CV drop and the Slaser is the laser area. In the experiment, the Ssub on the Si wafer was 1.2 times larger than that on the Ag NPs30-Ag20- AAO SERS substrate. In the SERS measurement, the diameter of the laser area was 1 µm. 2 Through calculation, the Slaser was 0.785 µm . Therefore, the value of Nbulk/NSERS was calculated as 0.833 × 104. Therefore, according to Equation (6), the EEF value of the Ag 6 NPs30-Ag20-AAO SERS substrate was 1.0083 × 10 . The EEF value is less than TEF value. In the simulation model of electromagnetic field intensity, the Ag nanospheres composed of silver atoms were closely constructed, and large-scale “hot spots” were stimulated in the narrow nanogaps. On the other hand, there was a monolayer Ag NPs film modified on the surface of the Ag-AAO model in Figure6c. However, in the experiment, Ag NPs were adsorbed in multiple layers. The aforementioned two main reasons caused the TEF value to be greater than the EEF value. So far, we have successfully detected 4- aminothiophenol, acephate, MB and CV molecules with the Ag NPs30-Ag20-AAO substrate. These results indicate the universality of the Ag NPs30-Ag20-AAO SERS substrate in the field of Raman spectroscopy.

3.7. Detection of the Baicalein by the Ag NPs30-Ag20-AAO Substrate

According to the above analysis, we know that the Ag NPs30-Ag20-AAO substrate has strong electromagnetic ﬁeld enhancement, ultra-high sensitivity and excellent Raman signal reproducibility. At a very low concentration of 10−10 mg/mL, acephate residue was successfully detected on the surface of Scutellaria baicalensis. In order to further study the application prospects of the substrate in the detection of real active ingredients in traditional Chinese medicine, we prepared Baicalein solutions with the concentration ranging from 1 ng/mL to 10 fg/mL. Scutellaria baicalensis is a kind of traditional Chinese medicine with roots as a medicine. It is mainly used for warm heat disease, upper respiratory tract infection, lung heat cough, pneumonia, dysentery, hypertension and so on [40]. Modern Chinese medicine Coatings 2021, 11, 1054 14 of 17

research showed that Baicalein was the main active ingredient of Scutellaria baicalensis [41]. Baicalein can reduce cerebrovascular resistance, improve cerebral blood circulation and increase cerebral blood ﬂow and anti platelet aggregation. It is used for the treatment of paralysis after cerebrovascular disease. Baicalein is an effective component in the body. Coatings 2021, 11, x FOR PEER REVIEWAfter Baicalein enters the animal body, it is rapidly transformed into Baicalin14 of 17 and other

metabolites in the blood.

Figure 10. 10. ((aa) )SERS SERS spectra spectra of of10− 102 M− 2CVM solution CV solution on the on Si thewafer Si; wafer; (b) SERS (b )spectra SERS spectraof 10−6 M of CV 10 −6 M CV solution on the Ag NPs30-Ag20-AAO SERS substrate. solution on the Ag NPs30-Ag20-AAO SERS substrate.

3.7. DetectionTherefore, of the a Baicalein reasonable, by the low-cost Ag NPs30 and-Ag20 sensitive-AAO Substrate method for the detection of Baicalein wasAccording essential. to Figure the above 11a analysis, shows the we Ramanknow that spectra the Ag ofNPs Baicalein30-Ag20-AAO at the substrate concentrations has of strong electromagnetic field enhancement, ultra-high sensitivity and excellent Raman sig- 1 ng/mL to 10 fg/mL on the Ag NPs30-Ag20-AAO substrate obtained by SERS technology. nal reproducibility. At a very low concentration of 10−10 mg/mL, acephate residue was Figure 11b1,b2 shows the Raman spectra of Baicalein powder and 1 ng/mL Baicalein. In successfully detected on the surface of Scutellaria baicalensis. In order to further study the these Raman spectra, the characteristic peaks of Baicalein, including 691 cm−1, 1063 cm−1, application prospects of the substrate in the detection of real active ingredients in tradi- 1370 cm−1 and 1599 cm−1, can be clearly observed. With the decrease of Baicalein concen- tional Chinese medicine, we prepared Baicalein solutions with the concentration ranging tration,from 1 ng/mL the intensities to 10 fg/mL. of the Raman characteristic peaks decreased. When the concentration was asScutellaria low as baicalensis 10 fg/mL, is thea kind characteristic of traditional peak Chinese can medicine still be clearly with roots identiﬁed. as a medi- Taking the −1 1599cine. It cm is mainlycharacteristic used for warm peak heat as andisease, example, upper when respiratory the logarithms tract infection, of Baicaleinlung heat concen- trationcough, pneumonia, and Raman dysentery, intensity werehypertension calculated and atso the on same[40]. Modern time, a Chinese perfect linearmedicine correlation curveresearch was showed shown that in Baicalein Figure was11c, the where main active the calculation ingredient of equation Scutellaria was baicalensisy = 6.181 [41].+ 0.197 x andBaicalein R2 was can 0.99128.reduce cere Thebrovascular practical resistance, detection improve results show cerebral that blood this circulation high-performance and Ag Coatings 2021, 11, x FOR PEER REVIEWincrease cerebral blood flow and anti platelet aggregation. It is used for the treatment of 15 of 17 NPs30-Ag20-AAO SERS substrate can be applied for the rapid and quantitative detection of otherparalysis label-free after cerebrovascular active ingredient disease. molecules Baicalein in is other an effective traditional component Chinese in medicines.the body. After Baicalein enters the animal body, it is rapidly transformed into Baicalin and other metabolites in the blood. Therefore, a reasonable, low-cost and sensitive method for the detection of Baicalein was essential. Figure 11a shows the Raman spectra of Baicalein at the concentrations of 1 ng/mL to 10 fg/mL on the Ag NPs30-Ag20-AAO substrate obtained by SERS technology. Figure 11b1,b2 shows the Raman spectra of Baicalein powder and 1 ng/mL Baicalein. In these Raman spectra, the characteristic peaks of Baicalein, including 691 cm−1, 1063 cm−1, 1370 cm−1 and 1599 cm−1, can be clearly observed. With the decrease of Baicalein concentration, the intensities of the Raman characteristic peaks decreased. When the concentration was as low as 10 fg/mL, the characteristic peak can still be clearly identified. Taking the 1599 cm−1 characteristic peak as an example, when the logarithms of Baicalein concentration and Raman intensity were calculated at the same time, a perfect linear correlation curve was shown in Figure 11c, where the calculation equation was y = 6.181 + 0.197 x and R2 was 0.99128. The practical detection results show that this high-performance Ag NPs30-

Figure 11. (a) Raman spectraAg of20 -BaicaleinAAO SERS at substratedifferent canconcentrations be applied for ranging the rapid from and 1 ng/mLquantitative to 10 detectionfg/mL; (b 1of,b other2) Raman Figure 11. (a) Raman spectra of Baicalein at different concentrations ranging from 1 ng/mL to 10 fg/mL; (b1,b2) Raman spectra of Baicalein powder labeland 1-free ng/mL active Baicalein; ingredient (c) correspondingmolecules in other calibration traditional curve Chinese of the peakmedicines. intensity at 1599 cm−1. spectra of Baicalein powder and 1 ng/mL Baicalein; (c) corresponding calibration curve of the peak intensity at 1599 cm−1.

as an inducer, the designed plasma nanoarray was realized by the strategy of the self- assembly of Ag NPs at the N-hexane/water interface. The close-packed Ag NPs on the Ag20-AAO nanoarray with the nanogaps less than 10 nm between the neighboring Ag NPs stimulated uniform and high intensity electromagnetic enhancement “hot spots”. The high-performance Ag NPs30-Ag20-AAO substrate was used to detect the residue of acephate on the surface of Scutellaria baicalensis, which proved that the substrate had excellent Raman signal reproducibility and reliable practicability. In the practical detection, the Raman spectra of Baicalein and its concentration dependence were given, and the LOD was located at 10 fg/mL. The superiority of the constructed Ag NPs30-Ag20-AAO SERS platform in the current study demonstrated the feasibility of both pesticide residues analysis and Chinese medicine active components detection. It indicated that this preparation method has great application potential in the trace detection of other active components of traditional Chinese medicine, pesticides and antibiotics.

Author Contributions: Conceptualization, M.W. and L.W.; methodology, L.W. and M.W.; software, J.G. (Jialin Ge) and W.Y.; validation, S.X., M.W. and J.G. (Jianjun Gu); formal analysis, C.C., K.L. and J.G. (Jungai Gu); investigation, J.G. (Jianjun Gu) and W.H.; resources, G.S. and S.X.; data curation, G.S. and Z.Z.; writing—original draft preparation, G.S., Z.Z. and W.Y.; writing—review and editing, G.S., C.C. and W.H.; visualization, J.G. (Jungai Gu) and J.G. (Jialin Ge); supervision, K.L.; project administration, G.S. and S.X.; funding acquisition, L.W., G.S. and S.X. All authors have read and agreed to the published version of the manuscript. Funding: This work was funded by the Science and Technology Project of Hebei Education Depart- ment (QN2021236), the foundation of Hebei Normal University for nationalities (NO. DR2018004) and the “Technology Innovation Guidance Project-Science and Technology Work Conference” of the Hebei Provincial Department of Science and Technology. Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Data Availability Statement: Data sharing is not applicable to this article. Conflicts of Interest: The authors declare no conflict of interest.

References 1. Ru, E.C.L.; Etchegoin, P.G. Single-molecule surface-enhanced Raman spectroscopy. Annu. Rev. Phys. Chem. 2012, 63, 65–87. 2. Zheng, X.S.; Jahn, I.J.; Weber, K.; Cialla-May, D.; Popp, J. Label-free SERS in biological and biomedical applications: Recent progress, current challenges and opportunities. Spectrochim. Acta A 2018, 197, 56–77.

Coatings 2021, 11, 1054 15 of 17

4. Conclusions In summary, a facile approach for the high technical mature fabrication of the 30 nm Ag NPs self-assembly Ag20-AAO nanoarray was successfully demonstrated for sensing pesticide residues and active components of traditional Chinese medicine. Using ethanol as an inducer, the designed plasma nanoarray was realized by the strategy of the self- assembly of Ag NPs at the N-hexane/water interface. The close-packed Ag NPs on the Ag20-AAO nanoarray with the nanogaps less than 10 nm between the neighboring Ag NPs stimulated uniform and high intensity electromagnetic enhancement “hot spots”. The high- performance Ag NPs30-Ag20-AAO substrate was used to detect the residue of acephate on the surface of Scutellaria baicalensis, which proved that the substrate had excellent Raman signal reproducibility and reliable practicability. In the practical detection, the Raman spectra of Baicalein and its concentration dependence were given, and the LOD was located at 10 fg/mL. The superiority of the constructed Ag NPs30-Ag20-AAO SERS platform in the current study demonstrated the feasibility of both pesticide residues analysis and Chinese medicine active components detection. It indicated that this preparation method has great application potential in the trace detection of other active components of traditional Chinese medicine, pesticides and antibiotics.

Author Contributions: Conceptualization, M.W. and L.W.; methodology, L.W. and M.W.; software, J.G. (Jialin Ge) and W.Y.; validation, S.X., M.W. and J.G. (Jianjun Gu); formal analysis, C.C., K.L. and J.G. (Jungai Gu); investigation, J.G. (Jianjun Gu) and W.H.; resources, G.S. and S.X.; data curation, G.S. and Z.Z.; writing—original draft preparation, G.S., Z.Z. and W.Y.; writing—review and editing, G.S., C.C. and W.H.; visualization, J.G. (Jungai Gu) and J.G. (Jialin Ge); supervision, K.L.; project administration, G.S. and S.X.; funding acquisition, L.W., G.S. and S.X. All authors have read and agreed to the published version of the manuscript. Funding: This work was funded by the Science and Technology Project of Hebei Education Depart- ment (QN2021236), the foundation of Hebei Normal University for nationalities (NO. DR2018004) and the “Technology Innovation Guidance Project-Science and Technology Work Conference” of the Hebei Provincial Department of Science and Technology. Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Data Availability Statement: Data sharing is not applicable to this article. Conﬂicts of Interest: The authors declare no conﬂict of interest.

References 1. Ru, E.C.L.; Etchegoin, P.G. Single-molecule surface-enhanced Raman spectroscopy. Annu. Rev. Phys. Chem. 2012, 63, 65–87. [PubMed] 2. Zheng, X.S.; Jahn, I.J.; Weber, K.; Cialla-May, D.; Popp, J. Label-free SERS in biological and biomedical applications: Recent progress, current challenges and opportunities. Spectrochim. Acta A 2018, 197, 56–77. [CrossRef] 3. Li, C.H.; Liu, A.H.; Zhang, C.; Wang, M.H.; Li, Z.; Xu, S.C.; Jing, S.Z.; Yu, J.; Yang, C.; Man, B.Y. Ag gyrus-nanostructure supported on graphene/Au ﬁlm with nanometer gap for ideal surface enhanced Raman scattering. Opt. Express 2017, 25, 20631–20641. [CrossRef][PubMed] 4. Yu, J.; Yang, M.; Li, Z.; Liu, C.; Wei, Y.; Zhang, C.; Man, B.; Lei, F. Hierarchical particle-in-quasicavity architecture for ultratrace in situ Raman sensing and its application in real-time monitoring of toxic pollutants. Anal. Chem. 2020, 92, 14754–14761. [CrossRef] 5. Zhao, Y.Y.; Qi, F.; Zhao, C.L.; Xie, C.X.; Wang, J.G.; Chang, C.T.; Li, Y.J.; Zhang, L.C. Facile fabrication of ultrathin freestanding nanoporous Cu and Cu-Ag ﬁlms with high SERS sensitivity by dealloying Mg-Cu(Ag)-Gd metallic glasses. J. Mater. Sci. Technol. 2021, 70, 205–213. [CrossRef] 6. Meng, S.; Chen, R.; Xie, J.; Li, J.; Cheng, J.; Xu, Y.; Cao, H.; Wu, X.; Zhang, Q.; Wang, H. Surface-enhanced Raman scattering holography chip for rapid, sensitive and multiplexed detection of human breast cancer-associated MicroRNAs in clinical samples. Biosens. Bioelectron. 2021, 190, 1113470–1113479. [CrossRef] 7. Fleischmann, M.; Hendra, P.J.; McQuillan, A.J. Raman spectra of pyridine adsorbed at a silver electrode. Chem. Phys. Lett. 1974, 26, 163–166. [CrossRef] 8. Cong, S.; Yuan, Y.; Chen, Z.; Hou, J.; Yang, M.; Su, Y.; Zhang, Y.; Li, L.; Li, Q.; Geng, F.; et al. Noble metal-comparable SERS enhancement from semiconducting metal oxides by making oxygen vacancies. Nat. Commun. 2015, 6, 7800–7806. [CrossRef] [PubMed] Coatings 2021, 11, 1054 16 of 17

9. Salomon, L.; Bassou, G.; Aourag, H.; Dufour, J.P.; De Fornel, F.; Carcenac, F.; Zayats, A.V. Local excitation of surface plasmon polaritons at discontinuities of a metal film: Theoretical analysis and optical near-field measurements. Phys. Rev. B 2002, 65, 125409. [CrossRef] 10. Willets, K.A.; Van Duyne, R.P. Localized surface plasmon resonance spectroscopy and sensing. Annu. Rev. Phys. Chem. 2007, 58, 267–297. [CrossRef] 11. Otto, A. Surface-enhanced Raman scattering: “Classical” and “Classical” Origins. In Light Scattering in Solids IV. Topics in Applied Physics; Springer: Berlin, Germany, 1984; Volume 54, pp. 289–418. 12. Campion, A.; Kambhampati, P. Surface enhanced Raman scattering. Chem. Soc. Rev. 1998, 27, 241–250. [CrossRef] 13. Wang, Y.X.; Liu, S.S.; Gao, W.T.; Zhang, Y.J.; Yang, J.H. Surface-enhanced Raman spectroscopy based on ordered nanocap arrays. Superlattice. Microst. 2012, 52, 750–758. [CrossRef] 14. Xu, H.; Jian, Q.; Xu, L.; Gao, S. Wireless battery-free generation of electric fields on one-dimensional asymmetric Au/ZnO nanorods for enhanced Raman sensing. Anal. Chem. 2021, 93, 9286−9295. [CrossRef] 15. Tang, L.; Tian, C.; Gu, W.; Jiang, Z. Surface-enhanced Raman scattering-based lateral flow immunoassay mediated by hydrophilic- hydrophobic Ag-modified PMMA substrate. Spectrochim. Acta A Mol. Biomol. Spectrosc. 2021, 262, 120092. [CrossRef] 16. Caldwell, J.; Taladriz-Blanco, P.; Rothen-Rutishauser, B.; Petri-Fink, A. Detection of sub-micro- and nanoplastic particles on gold nanoparticle-based substrates through surface-enhanced Raman scattering (SERS) Spectroscopy. Nanomaterials 2021, 11, 1149. [CrossRef][PubMed] 17. Zhang, P.; Liu, G.Q.; Feng, S.J.; Zhao, X. Engineering of flexible granular Au nanocap ordered array and its surface enhanced Raman spectroscopy effect. Nanotechnology 2020, 31, 035303–035312. [CrossRef][PubMed] 18. Ding, Q.Q.; Kang, Y.L.; Li, W.L.; Sun, G.F.; Liu, H.; Li, M.; Ye, Z.R.; Zhou, M.; Zhou, J.G.; Yang, S.K. Bioinspired brochosomes as broadband and omnidirectional surface-enhanced Raman scattering substrates. J. Phys. Chem. Lett. 2019, 10, 6484–6491. [CrossRef] 19. Zhang, X.; Zhang, X.; Luo, C.L.; Liu, Z.Q.; Chen, Y.Y.; Dong, S.L.; Jiang, C.Z.; Yang, S.K.; Wang, F.B.; Xiao, X.H. Volume-enhanced Raman scattering detection of viruses. Small 2019, 15, 1805516–1805523. [CrossRef] 20. Politano, G.G.; Cazzanelli, E.; Versace, C.; Vena, C.; De Santo, M.P.; Castriota, M.; Ciuchi, F.; Bartolino, R. Graphene oxide on magnetron sputtered silver thin films for SERS and metamaterial applications. Appl. Surf. Sci. 2018, 427, 927–933. [CrossRef] 21. Yilmaz, M. Silver-nanoparticle-decorated gold nanorod arrays via bioinspired polydopamine coating as surface-enhanced Raman spectroscopy (SERS) platforms. Coatings 2019, 9, 198. [CrossRef] 22. Demirci, G.; Muszynska, J.; Cetinkaya, O.; Filipczak, P.; Zhang, Y.M.; Nowaczyk, G.; Halagan, K.; Ulanski, J.; Matyjaszewski, K.; Pietrasik, J.; et al. Effective SERS materials by loading Ag nanoparticles into poly(acrylic acid-stat-acrylamide)-block-polystyrene nano-objects prepared by PISA. Polymer 2021, 224, 123747–123756. [CrossRef] 23. Guo, L.T.; Cao, H.W.; Cao, L.P.; Li, N.; Zhang, A.Q.; Shang, Z.B.; Jiao, T.F.; Liu, H.L.; Wang, M.L. Improve optical properties by modifying Ag nanoparticles on a razor clam SERS substrate. Opt. Express 2021, 29, 5152–5165. [CrossRef][PubMed] 24. Shi, G.C.; Wang, M.L.; Zhu, Y.Y.; Wang, Y.H.; Xu, H.J. A novel natural SERS system for crystal violet detection based on graphene oxide wrapped Ag micro-islands substrate fabricated from Lotus leaf as a template. Appl. Surf. Sci. 2018, 459, 802–811. [CrossRef] 25. Yang, W.; Li, Z.; Lu, Z.Y.; Yu, J.; Huo, Y.Y.; Man, B.Y.; Pan, J.; Si, H.P.; Jiang, S.Z.; Zhang, C. Graphene-Ag nanoparticles-cicada wings hybrid system for obvious SERS performance and DNA molecular detection. Opt. Express 2019, 27, 3000–3013. [CrossRef] 26. Fang, Y.; Seong, N.H.; Dlott, D.D. Measurement of the distribution of site enhancements in surfaceenhanced Raman scattering. Science 2008, 321, 388–392. [CrossRef] 27. Yan, X.Y.; Wang, M.Y.; Sun, X.; Wang, Y.H.; Shi, G.C.; Ma, W.L.; Hou, P. Sandwich-like Ag@Cu@CW SERS substrate with tunable nanogaps and component based on the Plasmonic nanonodule structures for sensitive detection crystal violet and 4-aminothiophenol. Appl. Surf. Sci. 2019, 479, 879–886. [CrossRef] 28. Lee, P.C.; Meisel, D.J. Adsorption and surface-enhanced Raman of dyes on silver and gold sols. J. Phys. Chem. B 1982, 86, 3391–3395. [CrossRef] 29. Fuchs, R. Theory of the optical properties of ionic crystal cubes. Phys. Rev. B 1975, 11, 1732–1740. [CrossRef] 30. Gai, H.F.; Wang, J.; Tian, Q. Modified Debye model parameters of metals applicable for broadband calculations. Appl. Optics 2007, 46, 2229–2233. [CrossRef] 31. Wang, M.L.; Yan, X.Y.; Shi, G.C.; Shang, Z.B.; Zhang, A.Q.; Ma, W.L. Optical properties of Ag@cicada wing substrate deposited by Ag nanoparticles. Curr. Appl. Phys. 2020, 20, 1253–1262. [CrossRef] 32. García-Vidal, F.J.; Pendry, J.B. Collective theory for surface enhanced Raman scattering. Phys. Rev. Lett. 1996, 77, 1163–1166. [CrossRef] 33. Muhammad, M.; Shao, C.S.; Huang, Q. Label-free SERS diagnostics of radiation-induced injury via detecting the biomarker Raman signal in the serum and urine bio-samples based on Au-NPs array substrates. Spectrochim. Acta A Mol. Biomol. Spectrosc. 2019, 233, 117282–117291. [CrossRef] 34. Wang, M.L.; Shang, Z.B.; Yan, X.Y.; Shi, G.C.; Cao, H.W.; Ma, W.L.; Jiao, T.F. Enhance fluorescence study of grating structure based on three kinds of optical disks. Opt. Commun. 2021, 481, 126522–126530. [CrossRef] 35. Li, C.Y.; Huang, Y.Q.; Lai, K.Q.; Rasco, B.A.; Fan, Y.X. Analysis of trace methylene blue in fish muscles using ultra-sensitive surface-enhanced Raman spectroscopy. Food Control 2016, 65, 99–105. [CrossRef] Coatings 2021, 11, 1054 17 of 17

36. Lin, S.; Lin, X.; Shang, Y.; Han, S.; Hasi, W.; Wang, L. Self-assembly of faceted gold nanocrystals for surface-enhanced Raman scattering application. J. Phys. Chem. C 2019, 123, 24714–24722. [CrossRef] 37. Parsons, H.M.; Ekman, D.R.; Collette, T.W.; Viant, M.R. Spectral relative standard deviation: A practical benchmark in metabolomics. Analyst 2009, 134, 478–485. [CrossRef] 38. Lv, M.Y.; Teng, H.Y.; Chen, Z.Y.; Zhao, Y.M.; Zhang, X.; Liu, L.; Wu, Z.L.; Liu, L.M.; Xu, H.J. Low-cost Au nanoparticle-decorated cicada wing as sensitive and recyclable substrates for surface enhanced Raman scattering. Sens. Actuators B. Chem. 2015, 209, 820–827. [CrossRef] 39. Zhang, M.F.; Meng, J.T.; Wang, D.P.; Tang, Q.; Chen, T.; Rong, S.Z.; Liu, J.Q.; Wu, Y.C. Biomimetic synthesis of hierarchical 3D Ag butterfly wing scale arrays/graphene composites as ultrasensitive SERS substrates for efficient trace chemical detection. J. Mater. Chem. C 2018, 6, 1933–1943. [CrossRef] 40. Chen, M.T.; Xiao, H.T.; Chen, B.; Bian, Z.X.; Kwan, H.Y. The advantages of using Scutellaria baicalensis and its flavonoids for the management of non-viral hepatocellular carcinoma. J. Funct. Foods 2021, 78, 104389–104401. [CrossRef] 41. Liao, H.F.; Ye, J.; Gao, L.L.; Liu, Y.L. The main bioactive compounds of Scutellaria baicalensis Georgi. for alleviation of inflammatory cytokines: A comprehensive review. Biomed. Pharmacother. 2021, 133, 110917–110933. [CrossRef][PubMed]