Au@Ag alloy containing interior hotspots galvanically prepared on an in-vitro diagnostic material for ultrasensitive and rapid detection of plasticizers

Au@Ag alloy containing interior hotspots galvanically prepared on an in-vitro diagnostic material for ultrasensitive and rapid detection of plasticizers | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Au@Ag alloy containing interior hotspots galvanically prepared on an in-vitro diagnostic material for ultrasensitive and rapid detection of plasticizers Soo Hyun Lee, ChaeWon Mun, Jun-Yeong Yang, Seunghun Lee, Sung-Gyu Park This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9024229/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 9 You are reading this latest preprint version Abstract The impacts of toxic environmental substances such as plasticizers on human health have been intensively studied in recent years. For ultrasensitive and reliable detection of plasticizers, in the present work, we developed a bimetallic surface-enhanced Raman spectroscopy (SERS) platform in which Ag in Ag-coated cotton fabric nanopillars were subjected to GR with Au (Au@Ag/CFNPs) in the presence of analyte molecules. The hollow regions, which originated from the imbalanced stoichiometric ratio between Au and Ag, functioned as the plasmonic interior hotspots and molecular pathways. The spontaneous galvanic reaction (GR), based on the intrinsic material properties ( i.e. , reduction potential), exhibited repeatable SERS signals with relative standard deviation values of < 10%. For the plasticizers such as benzyl butyl phthalate (BBP) and bisphenol A (BPA), the proposed platform demonstrated sub-ppm sensitivity and a linear relationship between signal intensity and analyte concentration. To investigate the feasibility of the proposed platform in practical applications, a test solution extracted from an actual plastic sample containing BPA was measured using the Au@Ag/CFNP platform. Based on an in-vitro diagnostic cotton fabric (CF) used as a base material, a lateral flow assay (LFA) kit templated with Ag/CFNPs was prepared. With the injection of BBP molecules and Au precursor, the SERS signals could be detected from the LFA-SERS kit down to a BBP concentration of 10 ppm. The results indicate that the Au@Ag/CFNP platform with GR-induced interior hotspots could serve as an alternative to existing standard techniques ( e.g. , pyrolysis, gas chromatography–mass spectrometry) for on-site early screening of plasticizers in food, daily products, and environments. Surface-enhanced Raman spectroscopy Galvanic reaction Interior hotspots Plasticizers Lateral flow assay Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 1. Introduction Plasticizers are indispensable additives that impart persistence, flexibility, and stability to plastics and epoxy resins during the manufacturing of products such as containers, bottles, and thermal papers. However, these compounds have emerged as environmental risk factors [ 1 , 2 ]. Plasticizers readily leach from plastics owing to their non-covalent interaction with polymers, and then migrate into foods, beverages, and the environment ( e.g. , water and soils) [ 3 ]. These compounds also accumulate in the human body via multiple exposure pathways, disrupting endocrine, developmental, immune, reproductive, and hormonal systems [ 4 – 6 ]. In Europe, the usage of plasticizers is restricted to concentrations > 10 3 ppm, as stated in the Restriction of Hazardous Substances (RoHS) 2 directive [ 7 ] and the Registration, Evaluation, Authorization, and Restriction of Chemicals (REACH) regulation [ 8 ]. One such compound of particular concern is benzyl butyl phthalate (BBP), which is used to increase the flexibility of polyvinyl chloride. Bisphenol A (BPA) is also included in the candidate list of substances of very high concern according to Article 59(10) of the REACH regulation since 2017, and its utilization in thermal papers is restricted (Regulation (EU) 1907/2006) with a maximum level of 0.02% by weight by Regulation (EU) 2016/2235, effective since 2 January 2020 [ 9 ]. Currently, plasticizers can be precisely identified and quantified using pyrolysis, gas chromatography–mass spectrometry (GC–MS), and high-performance liquid chromatography (HPLC) analyses. However, the high cost, complexity, and low throughput of these systems remain challenges in practical industrial fields. Therefore, the development of an ultrasensitive, rapid, and accurate sensing platform would be of great benefit. Surface-enhanced Raman spectroscopy (SERS) has been widely used to identify individual compounds because energy differences between incident and scattered light ( i.e. , inelastic scattering) directly correspond to the vibrational states of molecules [ 10 , 11 ]. SERS signals are predominantly amplified by electromagnetic ‘hotspots’—intense electric fields ( E -fields) confined in dielectric media between noble metal nanostructures ( e.g. , Au, Ag, and Cu)—a phenomenon referred to as localized surface plasmon resonance (LSPR) [ 12 , 13 ]. Because the strength of E -fields is inversely and exponentially related to the gap distance, various top-down and bottom-up approaches have been attempted to fabricate SERS platforms with narrow hotspots (typically ≤ 10 nm) [ 14 – 17 ]. Despite SERS having single-molecule sensitivity without the need for a labeling process, SERS is not used as a standard analytic method because i) there is the trade-off between enhancement factor and technical simplicity; ii) Brownian motion drives a portion of molecules to be adsorbed at the plasmonic active regions; and iii) there is a scale mismatch between hotspots and analytes, especially biomaterials. To overcome these issues, convergent electrochemistry-SERS (EC-SERS) platforms exploiting interior hotspots have been extensively studied in recent years [ 18 – 20 ]. EC-SERS techniques can achieve superior sensitivity due to i) the active participation of molecules, even on a large scale; ii) the match between the dimensions of the field confinement domain and the target analyte, and iii) the coupling effect between interior and conventional (herein referred to as environmental) hotspots. Interior hotspots are realized by encapsulating analyte molecules with plasmonic materials via electrodeposition [ 18 ], catalytic reaction [ 19 ], or galvanic reaction (GR) [ 20 ]. In particular, the GR method provides rapid, facile, and spontaneous redox processes between two or more noble metals based on differences in reduction potential ( E o ), and enables the formation of bimetallic and/or trimetallic compositions [ 21 – 23 ]. Au@Ag alloy is considered a promising candidate for EC-SERS platforms because i) the chemical inertness of Au protects the alloy against oxidation; ii) the compositional effect of hierarchical bimetals enhances plasmonic properties compared with single-metallic nanostructures [ 24 , 25 ]; iii) the unequal stoichiometry of Au and Ag leads to the creation of internal hollow regions through the Kirkendall effect [ 26 ], and these regions can potentially operate as plasmonic hotspots; and iv) molecules have an opportunity to penetrate into bimetallic layers during the GR process [ 26 ]. An additional advantage of Au@Ag alloys is that they can be formed via GR at room temperature without additional heating or catalysts, providing excellent reproducibility. Therefore, comprehensive studies on the characteristics and applications of interior hotspots in Au@Ag bimetallic platforms are required. In the present study, we developed an Au@Ag material with interior hotspots by galvanically replacing the Ag in Ag-coated cotton fabric nanopillars with Au (Au@Ag/CFNPs), and applied the resulting platform to the label-free detection of two plasticizers ( i.e. , BBP and BPA). The supporting nanostructures were directly defined on in-vitro diagnostic (IVD) cotton fabric (CF) through a maskless plasma etching process. The sacrificial Ag layer was rendered on the CFNPs (Ag/CFNPs) using the mirror reaction (MR), and then the Au@Ag bimetal was synthesized via the GR in the presence of analyte molecules. The presence and function of interior hotspots within the Au@Ag/CFNPs were experimentally and theoretically verified. The repeatability and sensitivity of the proposed platform were estimated for individual plasticizers, and these reference data were used to evaluate the sensing performance of the platform when applied to an actual plastic sample. A lateral flow assay (LFA) kit templated with an Ag/CFNP test line was designed for convenient detection of plasticizers. 2. Experimental section 2.1. Materials Silver nitrate (AgNO 3 ), potassium hydroxide (KOH), D-(+)-glucose, gold chloride trihydrate (HAuCl 4 ·3H 2 O), methylene blue (MB), BBP, and BPA were purchased from Sigma-Aldrich (St. Louis, MO, USA). Ammonia solution (NH 3 , 28%) was purchased from Junsei (Tokyo, Japan). Anhydrous ethyl alcohol (99.9%) was purchased from Samchun (Gangnam, South Korea). Sample pads (319 grade) consisting of CFs were obtained from Ahlstrom (Helsinki, Finland). 2.2. Fabrication of the Au@Ag/CFNP platform The supporting CFNPs were fabricated using a maskless plasma etching process. CF pads were loaded into a reactive-ion etching chamber, and then the base pressure was set to 7.2 mTorr. Under an O 2 flow rate of 52 sccm and a radio-frequency power of 100 W (13.56 MHz), O 2 plasma treatment was applied to the substrates for 2 min. Au@Ag/CFNPs were prepared via a two-step chemical process: Ag MR followed by GR. First, Tollens’ reagent containing 0.5 M AgNO 3 (5 mL), 0.8 M KOH (660 µL), and NH 3 (680 µL) was applied to the CFNPs, followed by injection of a 0.5 M glucose solution (660 µL). A sacrificial Ag layer formed on the CFNPs during the 10-min MR process. The resulting Ag/CFNP pads were rinsed with de-ionized water and then dried under ambient conditions. The as-prepared pads were cut into 5 mm × 5 mm squares. To generate interior hotspots, an in-situ GR process was applied by adding a 0.5 mM HAuCl 4 solution to the Ag/CFNPs in the presence of the solution to be tested. 2.3. SERS measurements An Ocean Optics portable probe spectrometer system (UQEPRO-Raman) was used to record real-time SERS spectra during the GR process. The samples were irradiated with a 785 nm laser with an optical power of 40 mW in a normal direction. SERS signals were collected for MB and plasticizers using acquisition times of 10 s and 30 s, respectively. 2.4. LFA-SERS assays To construct the LFA-SERS kit, a sample pad of dimensions 4.5 mm × 500 mm was prepared. The Ag/CFNPs were fabricated with an area of 4.5 mm × 30 mm to be used as a test line. The as-prepared pads were mounted on an adhesive backing membrane. A mixture (100 µL) of 0.5 mM HAuCl4 and test solution with a ratio of 1:1 was injected into the kit. After incubation for 2 min, SERS signals were measured from the plasmonic test line. 2.5. Theoretical computations The E -field profiles of the Ag/CFNP ( i.e. , GR time of 0 s) and Au@Ag/CFNP ( i.e. , GR time of 60 s) platforms were calculated numerically with the finite-difference time domain (FDTD) method using a commercial program (Ansys Lumerical 2021 R1.2). The structural parameters were extracted from scanning transmission electron microscopy (STEM) analyses. For the Ag/CFNPs, a conical CFNP (radius 90.9 nm, height 285.7 nm) was placed in a cuboid Ag layer (372.6 nm × 372.6 nm × 285.7 nm). The Ag head (radius 117.8 nm, height 90.9 nm) was located at the top of the CFNP. For the Au@Ag/CFNPs, void areas were constructed between the CFNP and Au@Ag shell. The bimetallic nanoporous layer was generated by mixing Au and Ag nanoparticles in a ratio of 1:9, and was applied to the cuboid layer (372.6 nm × 372.6 nm × 285.7 nm) and shell (radius 126.3 nm, height 109.0 nm). Assuming highly symmetric nanostructures, periodic boundary conditions were applied to the x - and y -axes, and a perfectly matched layer was used at the top and bottom layers. An x -polarized plane wave (785 nm) was normally incident on the designed structures. The mesh scale was fixed at 0.5 nm. For the optical parameters at 785 nm, the dielectric constants of Au and Ag were used, specifically ε Au = − 21.64 + i 0.74 and ε Ag = − 30.87 + i 2.99, respectively. The refractive indices of the CF pad and background water were set to 1.55 and 1.33, respectively. 2.6. Characterizations The morphological properties of the Ag/CFNPs and Au@Ag/CFNPs were observed by field-emission scanning electron microscopy (FE-SEM; JSM-6700F, Jeol, Tokyo, Japan) and STEM (JEM-ARM200F, Jeol, Tokyo, Japan). 3. Results and discussion 3.1. Strategy of the Au@Ag/CFNPs for detection of plasticizers The characteristics of plasmonic hotspots ( i.e. , scale, density, and occupancy rate) directly influence the SERS detection performance. A higher hotspot density increases the probability of molecules being located in narrower plasmonic active regions, making nanoporous platforms desirable for SERS applications [ 27 , 28 ]. In this study, the GR process coupled with the Kirkendall effect was used to create hollow regions in bimetallic nanostructures. The fabrication procedure of the proposed platform is shown in Scheme 1 . For practical applications, the sample pad, an IVD CF, was selected as a base substrate. To define the supporting CFNPs, O 2 plasma was applied to the pads (Scheme 1 (a)). After reaction with oxygen plasma, polar dangling bonds (resulting from chain breakage) and hydroxyl groups became the predominant active sites onto which Ag ions were adsorbed for seed formation [ 29 ]. An electroless MR process was employed to deposit the sacrificial Ag layer on the CFNPs (Scheme 1 (b)). The Ag layer was synthesized based on the redox process between Tollens’ reagent and glucose according to the equation: $${2\left[{\text{A}\text{g}\left({\text{N}\text{H}}_{3}\right)}_{2}\right]}^{+}+{\text{C}}_{5}{\text{H}}_{11}{\text{O}}_{5}\text{C}\text{H}\text{O}+{2\text{O}\text{H}}^{-}\to2\text{A}\text{g}+{\text{C}}_{5}{\text{H}}_{11}{\text{O}}_{5}\text{C}\text{O}\text{O}\text{H}+{\text{H}}_{2}\text{O}+4{\text{N}\text{H}}_{3}$$ 1 . Next, the GR process proceeded by injecting a HAuCl 4 solution onto the Ag/CFNPs (Scheme 1 (c)). The GR is a spontaneous reaction between two or more noble metals driven by differences in E o . The standard E o values of AuCl 4 − /Au and Ag + /Ag are approximately 1.5 V and 0.8 V versus the standard hydrogen electrode, respectively [ 30 , 31 ]. Because the material with higher standard E o accepts electrons ( i.e. , reduction) from the one with lower standard E o ( i.e. , oxidation), the Au@Ag bimetal is constructed by the following stoichiometric equation: $${3\text{A}\text{g}}_{\left(s\right)}+{{{\text{A}\text{u}\text{C}\text{l}}_{4}}^{-}}_{\left(aq\right)}\to3{{\text{A}\text{g}}^{+}}_{\left(aq\right)}+{\text{A}\text{u}}_{\left(s\right)}+{4{\text{C}\text{l}}^{-}}_{\left(aq\right)}$$ 2 . The diffusion rates of Au ( i.e. , inward direction) and Ag ( i.e. , outward direction) are not balanced because of the atomic exchange ratio of Au:Ag = 1:3 ( i.e. , the Kirkendall effect), and this imbalance contributes to the creation of hollow regions in the sacrificial layer. Such spaces play a dual role as plasmonic interior hotspots and molecular diffusion pathways. During the in-situ GR process, in which the test solution is present during hotspot formation, analyte molecules can be embedded in the hollow regions, possibly leading to a massive enhancement of their SERS signals. The proposed LFA-SERS kit, including a plasmonic membrane (herein, the IVD sample pad), exploits this signal amplification to enable the convenient detection of toxic and hazardous substances. 3.2. Morphological and optical characteristics of the Au@Ag/CFNPs via the GR process During Au@Ag/CFNP formation via the GR process, the following phenomena are typically observed: i) the creation of hollow regions due to the Kirkendall effect, ii) conversion of Ag to Au@Ag bimetal, and iii) re-reduction of a portion of oxidized Ag ions [ 32 , 33 ]. Therefore, the entire structural domain rapidly increases with processing time due to the formation of outer bimetallic layers [ 34 ]. This behavior was confirmed by examining the morphological changes of the Au@Ag/CFNPs at different GR times. Herein, the Ag/CFNPs were prepared by applying the MR (0.5 M AgNO 3 solution for 10 min) to the CFNPs (O 2 plasma treatment for 2 min), leading to the formation of an uniformly distributed Ag layer (Fig. 1 (a)). At a GR time of 30 s, partial Ag dissociation was observed (Fig. 1 (b)). For longer GR times, bimetallic layers containing a large number of hollow sites grew, and eventually covered the underlying structures (Figs. 1 (c)–(d)). Our previous research established that the signal enhancement in EC-SERS relies on the coupling of interior hotspots with environmental hotspots [ 18 , 26 , 35 ]. Therefore, we optimized the supporting Ag/CFNP platform to achieve maximum efficiency. Under plasma treatment, the polymeric nanostructures are generated by the following surface dynamics: selective etching, surface migration, agglomeration, and coalescence [ 34 , 36 , 37 ]. In this work, the CFNPs became agglomerated 3 min after the application of O 2 plasma to the CF substrate (Fig. S1 ). Given the morphological profile of the CFNPs ( i.e. , density and aspect ratio), the optimum O 2 plasma treatment time was determined to be 2 min. Next, the influence of MR parameters ( i.e. , synthesis time and AgNO 3 concentration) on the optical signal enhancement was investigated. To evaluate the SERS activities of the Au@Ag/CFNPs, herein, MB Raman dyes were used. During the MR process with 0.5 M AgNO 3 , Ag seeds were formed during the initial stage and then grew with synthesis time (Figs. S2(a)−(d)). The most intense MB signal was achieved from the Ag/CFNPs synthesized using an MR time of 10 min (Figs. S2(e)−(f)). At earlier times ( i.e. , 1 and 5 min), the plasmonic resonance was decreased due to the lower density of Ag nanostructures, while at later stages ( i.e. , longer than 10 min), the plasmonic resonance was degraded by significant structural inhomogeneity ( i.e. , non-uniformity). Experiments on Ag/CFNPs generated using different AgNO 3 concentrations showed that the optimum concentration was 0.5 M (Fig. S3). To optimize the GR process, experiments were performed in which the HAuCl 4 concentration ( i.e. , replacement ratio) was varied. The SERS signals showed the highest intensity at a Au precursor concentration of 0.5 mM (Fig. S4). Using the optimum conditions, the time evolution of the SERS signal at 1612 cm − 1 during the GR process in which Au@Ag/CFNPs were synthesized in the presence of MB (MB − Au@Ag/CFNPs) was measured (Fig. 1 (e)). The peak intensity reached saturation 40 s after injection of the Au precursor. To ensure reliable analysis, the optimum GR time was set to 60 s. To verify the superior function of interior hotspots, SERS spectra collected from three systems were compared: Ag/CFNPs to which MB was added (MB–Ag/CFNPs), in-situ MB–Au@Ag/CFNPs, and Au@Ag/CFNPs to which MB was added after Au@Ag/CFNP synthesis ( i.e. , post-addition) (Fig. 1 (f)). The spectrum obtained from the MB − Ag/CFNPs showed negligible SERS signals ( I ~ 20) because the MB molecules moving via Brownian motion were barely adsorbed onto the environmental hotspots ( i.e. , a low occupancy rate). When the dye was added to the already formed Au@Ag/CFNPs, SERS peaks were observed but with low intensity ( I ~ 1.8k). Although both interior and environmental hotspots were present in this case, delivery of MB molecules into the narrow interstitials was difficult due to the high surface tension of the solvent. In the case of the MB–Au@Ag/CFNPs formed during in-situ GR, by contrast, the dyes had the opportunity to penetrate into the desired areas during the dynamic atomic exchange that occurs in the GR, resulting in a remarkable amplification of the optical signals ( I ~ 60.3k). Accordingly, the SERS intensity for the in-situ MB − Au@Ag/CFNPs was approximately 33.5-fold higher than that of the post-addition of MB to Au@Ag/CFNPs. These results indicate that not only the plasmonic performance but also the molecular distribution play a crucial role in determining sensing efficiency of SERS platforms. 3.3. Investigation of the GR-induced interior hotspots in Au@Ag/CFNPs To observe the generation of the internal hollow regions in the Au@Ag/CFNPs, STEM analyses were conducted on the Ag/CFNPs and the Au@Ag/CFNPs formed after a GR time of 60 s (Figs. 2 (a)−(b)). The STEM image of the Ag/CFNPs shows a solid Ag layer on the supporting CFNPs. In this system, environmental hotspots are only provided by the transverse mode between neighboring Ag heads. The STEM image of the Au@Ag/CFNPs, by contrast, shows hollow regions with dimensions of 6–11 nm within the bimetallic layer, where the formation of the hollow regions is attributed to the Kirkendall effect. To confirm the formation of a bimetallic layer, elemental mapping analyses were performed (Fig. S5). After the GR process, the pure Ag component was transformed to Au@Ag alloys with an atomic percentage ratio of ~ 9.7:90.3. Au and Ag are miscible because they belong to the same space group ( i.e. , cubic Fm̅ 3 m ) and their lattice constants are similar (Au: 4.079 Å and Ag: 4.086 Å) [ 22 , 38]. In the high-resolution TEM image (Fig. 2 (c)), lattice fringes with a d -spacing of 0.24 nm, corresponding to the (111) planes of Au and Ag, are observed, demonstrating the highly crystalline nature of Au@Ag/CFNPs [29]. In addition, selected area electron diffraction analysis showed patterns consistent with a face-centered cubic structure [ 39 , 40], as shown in the inset of Fig. 2 (c). To theoretically verify the activation of interior hotspots, the E -field profiles of Ag/CFNPs and Au@Ag/CFNPs formed after a GR time of 60 s were calculated using the FDTD method (Fig. 2 (d)). The simulation models were constructed using the geometric parameters extracted from the STEM images. The following assumptions were made to simplify the structures: i) the Ag/CFNPs are periodically arranged, ii) the Ag layers are filled in the space between the CFNPs, iii) the Ag heads are hemispheres, and iv) the bimetallic layer is comprised of a 1:9 mixture of Au and Ag nanoparticles according to the elemental mapping result. For the Ag/CFNPs, weak E -fields were produced between the Ag heads because the heads are separated by a large gap and the strength of E -fields rapidly diminishes with gap distance ( i.e. , LSPR). After the GR, the field intensity became stronger because i) bimetallic layers containing internal hollow regions were generated and ii) the growth of the entire structural domain reduced the gap between the Au@Ag heads. Resonance coupling between the interior and environmental hotspots may further enhance molecular signals. According to the fourth power approximation, the theoretical enhancement factor of the Au@Ag/CFNPs was estimated to be 3.7 × 10 7 , which was much greater than that of the Ag/CFNPs (1.2 × 10 3 ). These results may explain the differences in spectral enhancement observed among the structural models in Fig. 1 (f). 3.4. SERS activities of in-situ MB − Au@Ag/CFNPs The SERS performance ( i.e. , repeatability and sensitivity) of the Au@Ag/CFNPs was investigated to assess the suitability of the platform for reliable molecular assays in practical settings. Using the optimum protocol, the SERS spectra of the Au@Ag/CFNPs formed in the presence of 100 nM MB dye solutions were collected from 20 different chips (Fig. 3 (a)). The SERS spectra from the 20 chips showed high consistency, and each spectrum showed features characteristic of MB. Herein, the MB peaks at 445, 772, 1390, and 1612 cm − 1 were selected for further analyses; the peak assignments of MB are summarized in Table S1 [ 41 , 42 ]. To calculate the relative standard deviation (RSD), the intensity profiles at the analytic peaks were extracted (Fig. 3 (b)). The RSD values were 6.46%, 9.14%, 7.13%, and 7.00% at 445, 772, 1390, and 1612 cm − 1 , respectively, indicating excellent signal uniformity. The excellent repeatability of the method can be attributed to i) the formation of plasmonic hotspots with high density and ii) the GR process being spontaneous and stable. For quantitative analyses, the signals from the Au@Ag/CFNPs formed in the presence of MB solutions with different concentrations ( n = 5) were measured (Fig. 3 (c)). Characteristic peaks of MB were observed in all of the spectra, and their intensities gradually diminished with decreasing concentration because of the reduction of the molecular adsorption probability described by the Langmuir model [ 43 ]. The proposed platform showed femtomolar sensitivity, demonstrating the superior plasmonic activities of the interior hotspots in the Au@Ag bimetals. To investigate the quantitative correlation between peak intensity and MB concentration, the intensity ( n = 5) of each analytic peak was plotted against concentration on a log-log scale (Fig. 3 (d)). For each peak, a highly linear relationship was observed with a correlation coefficient (R 2 ) of 0.99. The limit-of-detection (LOD) was also calculated using the equation $$\text{L}\text{O}\text{D}=\frac{3\times{S}_{\text{b}}}{m}$$ 3 , where S b is the standard deviation of blank measurements ( n = 5) at the analytic peak and m is the slope of the calibration curve. The average LOD value for the in-situ MB–Au@Ag/CFNPs was estimated to be 184.2 aM. As a result, the proposed Au@Ag/CFNP platform, which has the advantages of narrow interstitials, large molecular participation, and stable reaction, is a promising technology for tracing concentrations of hazardous substances. 3.5. Sensitivity evaluation of in-situ plasticizer − Au@Ag/CFNPs According to the RoHS 2 directive and REACH regulation, plasticizer concentrations must not exceed 10 3 ppm in manufactured plastic products. Therefore, detection systems must have a sensitivity below this level to enable reliable analysis of actual samples. In this work, the BBP and BPA were chosen as the analytic substances owing to their widespread usage in the manufacturing of plastics and epoxy resins [ 6 ]. Before the sensitivity evaluation, the activation of interior hotspots containing embedded plasticizers was investigated by comparing the SERS spectra of plasticizer − Ag/CFNP and in-situ plasticizer − Au@Ag/CFNP systems (Fig. S6), where the plasticizer concentration was set to 5000 ppm. For both plasticizers, negligible SERS signals were obtained from the Ag/CFNPs, whereas their signals were clearly observed after the in-situ GR process was applied. These results indicated that the lipophilic plasticizer molecules were able to penetrate into the narrow hollow regions effectively. The sensitivity of the proposed platform with GR-induced interior hotspots was evaluated for the detection of BBP and BPA. For the BBP–Au@Ag/CFNP system ( n = 5), representative SERS peaks at 646, 997, 1033, 1598, and 1725 cm − 1 were observed in the concentration range from 5 × 10 − 2 to 5 × 10 4 ppm (Fig. 4 (a)). The peak assignments of BBP, consisting of an aryl alkyl ester of phthalic acid, are presented in detail in Table S2 [ 44 , 45 ]. Among them, the peaks at 997 and 1033 cm − 1 were selected as analytic criteria; these peaks correspond to C − C in-plane bending in the secondary benzene ring and aromatic ring breathing in the primary benzene ring of BBP. From the logarithmic calibration curve, the R 2 and average LOD values were calculated to be ≥ 0.97 and 15.6 ppb, respectively (Fig. 4 (b)). In the case of BPA ( n = 5), the spectral characteristic features of the compound were detectable down to a concentration of 0.5 ppm (Fig. 4 (c)). The peak assignments of BPA, which contains a diphenylmethane bearing two hydroxyphenyl groups in the para positions, are summarized in Table S3 [ 46 , 47 ]. Plots of the intensities at 642 and 877 cm − 1 versus BPA concentration reveal a highly linear correlation, with R 2 values of ≥ 0.95 (Fig. 4 (d)). Based on the three standard deviation equation, the average LOD was found to be 219.7 ppb. These results indicate that the proposed platform is a viable alternative to current standard techniques ( e.g. , pyrolysis, GC–MS, and HPLC) for practical assays. 3.6. Detection of actual samples based on the in-situ Au@Ag/CFNP platform To demonstrate the practical applicability of the proposed platform, the system was applied to plasticizer detection in a polycarbonate (PC) plastic product. As reported in our previous study [ 34 ], the following protocol was employed to obtain the test solution: i) the PC product was cut into small pieces, ii) 0.3 g of the sample was immersed in ethanol, iii) the solution was left at room temperature for 2 h to extract plasticizers, and iv) the filtration was applied to remove plastic products [ 34 ]. During the extraction, no heating or pressure was applied. From the HPLC analysis, the plasticizer in the leaching solution was found to BPA with a concentration of ~ 10 ppm. The SERS spectrum of the test sample matched the representative spectrum of BPA with an excellent signal-to-noise ratio (Fig. 5 (a)). This was attributed to i) negligible background noise originating from the Au@Ag/CFNPs, ii) the boosting of optical signals by the GR-induced interior hotspots, and iii) the diffusion of unwanted large molecules to the bottom area of the CF substrate via pores. Because the plastic extraction process generates a leaching solution containing other compounds in addition to BPA, the BPA molecules must compete with these other compounds for access to the plasmonic hotspots. As a result, the spectrum of the leaching solution exhibited slightly lower intensity than the reference spectrum ( i.e. , 10 ppm BPA dissolved in ethanol). For example, the intensity at 877 cm − 1 of the leaching solution was close to the fitting line of the quantitative reference data (Fig. 5 (b)). Therefore, the proposed platform demonstrated great potential for detecting very low levels of toxic substances leached from plastic products, which is important for early screening in industrial applications. 3.7. LFA application For the on-site detection of plasticizers, convenient, simple, and cost-effective plasmonic sensing chips are required. For this purpose, a prototype LFA-SERS kit based on a Ag/CFNP strip was prepared (Fig. 6 (a)). The assembly of the kit was facilitated by the formation of plasmonic nanostructures on the IVD material ( i.e. , CFs). The LFA kit was designed with three compartments: i) a sample pad consisting of pristine CFs, ii) a Ag/CFNP strip of length 3 cm as an optical test line, and iii) an absorption pad. In the kit, the sample pad served as a filter to prevent large interferents ( e.g. , microplastics) reaching the test line. For sample loading, the plasticizer solution was mixed with a 0.5 mM HAuCl 4 solution at a volume ratio of 1:1, and a 100 µL droplet was applied to the sample pad. The mixture solution was driven by the capillary force. To ensure the formation of interior hotspots, the measurement was conducted 2 min after sample loading. The prototype LFA-SERS kit was tested using solutions with various BBP concentrations. Spectra collected from the kit ( n = 3) including the Au@Ag/CFNPs embedded with BBP are shown in Fig. 6 (b). The BBP molecules were successfully detected in the concentration range of 10 1 −10 4 ppm. Notably, the loaded samples covered both the internal and external surfaces, leading to initiation of the GR. For comparison with reference data, the intensity profiles at 997 and 1033 cm − 1 are displayed in Fig. 6 (c). The intensity levels in the LFA-SERS spectrum are lower than those in the reference spectrum because not all of the target molecules and Au precursors were involved in the formation of interior hotspots ( i.e. , component loss). From the logarithmic calibration, the R 2 and average LOD values were found to be ~ 0.90 and 1.4 ppm, respectively. Although the LFA-SERS kit showed lower detection performance compared with the reference data, it remains a rapid, sensitive, and high-throughput diagnostic tool for the early screening of plasticizers in plastic products. 4. Conclusions We reported the development of bimetallic nanostructures templated with interior hotspots on an IVD material ( i.e. , Au@Ag/CFNPs) for the ultrasensitive and on-site SERS detection of plasticizers. The plasmonic materials were prepared via a two-step chemical synthesis: Ag MR followed by GR. The internal hollow regions ( e.g. , interstitials, cracks, and voids) induced by the Kirkendall effect played a critical role as plasmonic hotspots and molecular pathways when the target analyte molecules were present during the process ( i.e. , in-situ GR). The activation of such dual-function interior hotspots was confirmed by comparison with structural models in which the target analyte molecules were added after the GR had completed. The enhancement factor of Au@Ag/CFNPs was calculated by FDTD simulation to be 3.7 × 10 7 . For MB dye, the Au@Ag/CFNP chips showed reliable sensing operation ( i.e. , RSDs <10%) with picomolar sensitivity. The proposed platform also detected the lipophilic plasticizers BBP and BPA at sub-ppm concentrations. The SERS spectrum of the leaching solution derived from a PC plastic product showed features characteristic of BPA without significant background noise. For convenient on-site diagnosis, an LFA kit templated with Ag/CFNPs was designed. Despite partial loss of the analytes and Au precursor during capillary flow into the sample pad, the LFA-SERS kit successfully traced the BBP in the concentration range of 10 1 −10 4 ppm. Therefore, the Au@Ag/CFNP platform with dual-function interior hotspots has great potential for detecting ecotoxicological substances in an ultrasensitive, rapid, and reliable manner, making it a promising technology for use in industrial applications. Declarations Declaration of Competing Interes ts The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Supplementary Information The online version contains supplementary material available at Acknowledgments This research was supported by the Fundamental Research Program (PNKB010) of the Korea Institute of Materials Science (KIMS), Republic of Korea. Author contributions Soo Hyun Lee: Analysis and interpretation of data, Investigation, Writing-Original draft preparation, Validation, Visualization. ChaeWon Mun: Investigation, Methodology. Jun-Yeong Yang: Investigation. Seunghun Lee: Investigation. Sung-Gyu Park: Conceptualization, Writing- Reviewing and Editing, Supervision, Project administration, Funding acquisition. Data availability The data that support the findings of this study are available from the corresponding author upon reasonable request. Ethics approval and consent to participate Not applicable. Consent for publication Not applicable. Competing interests The authors declare no competing interests. Clinical Trial Number Clinical trial number: not applicable. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-9024229","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":619132474,"identity":"fbe96423-c88d-45ae-88ed-9cebb9fdae63","order_by":0,"name":"Soo Hyun Lee","email":"","orcid":"","institution":"Korea Institute of Materials Science (KIMS)","correspondingAuthor":false,"prefix":"","firstName":"Soo","middleName":"Hyun","lastName":"Lee","suffix":""},{"id":619132475,"identity":"97e35a48-ebd5-421e-a201-ddca13783369","order_by":1,"name":"ChaeWon Mun","email":"","orcid":"","institution":"Korea Institute of Materials Science (KIMS)","correspondingAuthor":false,"prefix":"","firstName":"ChaeWon","middleName":"","lastName":"Mun","suffix":""},{"id":619132476,"identity":"8617de8f-eba3-4078-885c-849c6f185ac3","order_by":2,"name":"Jun-Yeong Yang","email":"","orcid":"","institution":"Korea Institute of Materials Science (KIMS)","correspondingAuthor":false,"prefix":"","firstName":"Jun-Yeong","middleName":"","lastName":"Yang","suffix":""},{"id":619132477,"identity":"5cf00fa7-b445-4c55-91dd-2863dff98f75","order_by":3,"name":"Seunghun Lee","email":"","orcid":"","institution":"Korea Institute of Materials Science (KIMS)","correspondingAuthor":false,"prefix":"","firstName":"Seunghun","middleName":"","lastName":"Lee","suffix":""},{"id":619132478,"identity":"0288c435-0226-4db5-b729-15fff365ce38","order_by":4,"name":"Sung-Gyu Park","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAyklEQVRIiWNgGAWjYNCCAzYGbFCmAbFa0kjXchiukrAW+f61Bz9XnDlvzCd2/AFzRQWDsXkDAS0GN94lS565cduMTTrHgPHMGQYzmQOEtEicMZBs+HDbBqiFgbGxjcFGgqDDZpwx/tnw4RxQS/oDxsZ/RGhhON9jJtlw4wDQYQkGjI0NDGYEtRjc4EuzbDiTbAzyy8GGYxLGhB3Wf/bwzYZjdobzZ6c/fNhQY2M4g6DDJHIQ7ANALkENDAz8Z4hQNApGwSgYBSMbAABTPT2ush8CRgAAAABJRU5ErkJggg==","orcid":"","institution":"Korea Institute of Materials Science (KIMS)","correspondingAuthor":true,"prefix":"","firstName":"Sung-Gyu","middleName":"","lastName":"Park","suffix":""}],"badges":[],"createdAt":"2026-03-03 23:53:14","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-9024229/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9024229/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":106868861,"identity":"85ddab36-d2b0-4fa7-a43c-3f5fd011bb39","added_by":"auto","created_at":"2026-04-14 09:34:16","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":519097,"visible":true,"origin":"","legend":"\u003cp\u003eMorphological properties of Ag/CFNPs and Au@Ag/CFNPs prepared using different GR times. SEM images of Au@Ag/CFNPs at GR times of (a) 0, (b) 30, (c) 60, and (d) 120 s. (e) Real-time MB signal at 1612 cm\u003csup\u003e–1\u003c/sup\u003e acquired from Au@Ag/CFNPs synthesized in the presence of MB (\u003cem\u003ein-situ\u003c/em\u003e MB–Au@Ag/CFNPs) and Ag/CFNPs to which MB was added (MB–Ag/CFNPs). (f) SERS spectra of MB–Ag/CFNPs, \u003cem\u003ein-situ\u003c/em\u003e MB–Au@Ag/CFNPs, and Au@Ag/CFNPs to which MB was added after Au@Ag/CFNP synthesis.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-9024229/v1/c17cf7b3f4bb6a28a8e01051.png"},{"id":106961437,"identity":"67a047d2-e34b-4bf0-a8ff-b57e50ab36bc","added_by":"auto","created_at":"2026-04-15 09:25:34","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":308362,"visible":true,"origin":"","legend":"\u003cp\u003eAnalysis of the interior hotspots. STEM images of (a) Ag/CFNPs and (b) Au@Ag/CFNPs. (c) High-resolution TEM and selected area electron diffraction images of Au@Ag/CFNPs. (d) Numerical calculation of the E-field distributions of Ag/CFNPs and Au@Ag/CFNPs using FDTD simulations.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-9024229/v1/d58bb1d444a0b5d29b6b2cb5.png"},{"id":106868863,"identity":"dde4f561-a932-41c7-b4fa-1201cc0a4d5e","added_by":"auto","created_at":"2026-04-14 09:34:16","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":509529,"visible":true,"origin":"","legend":"\u003cp\u003eSee image above for figure legend.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-9024229/v1/0913731238274ee5236c103a.png"},{"id":106961525,"identity":"c025da94-dde3-481b-b57c-56decd3c04d6","added_by":"auto","created_at":"2026-04-15 09:25:54","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":362079,"visible":true,"origin":"","legend":"\u003cp\u003eSee image above for figure legend.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-9024229/v1/227748cfc658f445feac0d80.png"},{"id":106868866,"identity":"5a6c28c5-13ba-491e-9d39-25b0b04a282b","added_by":"auto","created_at":"2026-04-14 09:34:16","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":120790,"visible":true,"origin":"","legend":"\u003cp\u003eFeasibility of the Au@Ag/CFNP platform for practical applications. (a) SERS spectrum of the solution extracted from a PC plastic compared with that of a reference solution (\u003cem\u003ei.e.\u003c/em\u003e, 10 ppm BPA dissolved in pure ethanol) and (b) the quantification based on the reference intensity at 887 cm\u003csup\u003e−1\u003c/sup\u003e.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-9024229/v1/e6d9f17d5e5c0e405041ea05.png"},{"id":106994132,"identity":"7fc47237-6d53-4504-9146-7d498f817dc8","added_by":"auto","created_at":"2026-04-15 15:04:49","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":229594,"visible":true,"origin":"","legend":"\u003cp\u003eLFA-SERS analyses. (a) Photographic image of the prototype LFA-SERS kit implanted with Au@Ag/CFNPs, (b) quantitative spectra of solutions containing different BBP concentrations acquired from LFA-SERS kits, and (c) their intensity profiles at 997 and 1033 cm\u003csup\u003e−1\u003c/sup\u003e compared with those of the reference BBP solutions.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-9024229/v1/b5e9190b0f8851b40eea09ff.png"},{"id":106994930,"identity":"67fde9f0-2f0c-4605-ab50-a77c194fe5af","added_by":"auto","created_at":"2026-04-15 15:20:39","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2755039,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9024229/v1/073e8f61-44eb-4c0c-a478-97a39971a8c9.pdf"},{"id":106960727,"identity":"1f253f1f-5078-44f2-a3b4-6e7d5d453eba","added_by":"auto","created_at":"2026-04-15 09:22:51","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":3645990,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementary.docx","url":"https://assets-eu.researchsquare.com/files/rs-9024229/v1/9afb414f8dd6a37ae591edbd.docx"},{"id":106961231,"identity":"2b122d46-b9f8-4f87-9aaf-0adbccdb0ab5","added_by":"auto","created_at":"2026-04-15 09:24:46","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":387295,"visible":true,"origin":"","legend":"","description":"","filename":"Scheme1.docx","url":"https://assets-eu.researchsquare.com/files/rs-9024229/v1/b1afe22b3b831f9fb030958a.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Au@Ag alloy containing interior hotspots galvanically prepared on an in-vitro diagnostic material for ultrasensitive and rapid detection of plasticizers","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003ePlasticizers are indispensable additives that impart persistence, flexibility, and stability to plastics and epoxy resins during the manufacturing of products such as containers, bottles, and thermal papers. However, these compounds have emerged as environmental risk factors [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Plasticizers readily leach from plastics owing to their non-covalent interaction with polymers, and then migrate into foods, beverages, and the environment (\u003cem\u003ee.g.\u003c/em\u003e, water and soils) [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. These compounds also accumulate in the human body \u003cem\u003evia\u003c/em\u003e multiple exposure pathways, disrupting endocrine, developmental, immune, reproductive, and hormonal systems [\u003cspan additionalcitationids=\"CR5\" citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. In Europe, the usage of plasticizers is restricted to concentrations\u0026thinsp;\u0026gt;\u0026thinsp;10\u003csup\u003e3\u003c/sup\u003e ppm, as stated in the Restriction of Hazardous Substances (RoHS) 2 directive [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e] and the Registration, Evaluation, Authorization, and Restriction of Chemicals (REACH) regulation [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. One such compound of particular concern is benzyl butyl phthalate (BBP), which is used to increase the flexibility of polyvinyl chloride. Bisphenol A (BPA) is also included in the candidate list of substances of very high concern according to Article 59(10) of the REACH regulation since 2017, and its utilization in thermal papers is restricted (Regulation (EU) 1907/2006) with a maximum level of 0.02% by weight by Regulation (EU) 2016/2235, effective since 2 January 2020 [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Currently, plasticizers can be precisely identified and quantified using pyrolysis, gas chromatography\u0026ndash;mass spectrometry (GC\u0026ndash;MS), and high-performance liquid chromatography (HPLC) analyses. However, the high cost, complexity, and low throughput of these systems remain challenges in practical industrial fields. Therefore, the development of an ultrasensitive, rapid, and accurate sensing platform would be of great benefit.\u003c/p\u003e \u003cp\u003eSurface-enhanced Raman spectroscopy (SERS) has been widely used to identify individual compounds because energy differences between incident and scattered light (\u003cem\u003ei.e.\u003c/em\u003e, inelastic scattering) directly correspond to the vibrational states of molecules [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. SERS signals are predominantly amplified by electromagnetic \u0026lsquo;hotspots\u0026rsquo;\u0026mdash;intense electric fields (\u003cem\u003eE\u003c/em\u003e-fields) confined in dielectric media between noble metal nanostructures (\u003cem\u003ee.g.\u003c/em\u003e, Au, Ag, and Cu)\u0026mdash;a phenomenon referred to as localized surface plasmon resonance (LSPR) [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Because the strength of \u003cem\u003eE\u003c/em\u003e-fields is inversely and exponentially related to the gap distance, various top-down and bottom-up approaches have been attempted to fabricate SERS platforms with narrow hotspots (typically\u0026thinsp;\u0026le;\u0026thinsp;10 nm) [\u003cspan additionalcitationids=\"CR15 CR16\" citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Despite SERS having single-molecule sensitivity without the need for a labeling process, SERS is not used as a standard analytic method because i) there is the trade-off between enhancement factor and technical simplicity; ii) Brownian motion drives a portion of molecules to be adsorbed at the plasmonic active regions; and iii) there is a scale mismatch between hotspots and analytes, especially biomaterials. To overcome these issues, convergent electrochemistry-SERS (EC-SERS) platforms exploiting interior hotspots have been extensively studied in recent years [\u003cspan additionalcitationids=\"CR19\" citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. EC-SERS techniques can achieve superior sensitivity due to i) the active participation of molecules, even on a large scale; ii) the match between the dimensions of the field confinement domain and the target analyte, and iii) the coupling effect between interior and conventional (herein referred to as environmental) hotspots. Interior hotspots are realized by encapsulating analyte molecules with plasmonic materials \u003cem\u003evia\u003c/em\u003e electrodeposition [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e], catalytic reaction [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e], or galvanic reaction (GR) [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn particular, the GR method provides rapid, facile, and spontaneous redox processes between two or more noble metals based on differences in reduction potential (\u003cem\u003eE\u003c/em\u003e\u003csup\u003eo\u003c/sup\u003e), and enables the formation of bimetallic and/or trimetallic compositions [\u003cspan additionalcitationids=\"CR22\" citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Au@Ag alloy is considered a promising candidate for EC-SERS platforms because i) the chemical inertness of Au protects the alloy against oxidation; ii) the compositional effect of hierarchical bimetals enhances plasmonic properties compared with single-metallic nanostructures [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]; iii) the unequal stoichiometry of Au and Ag leads to the creation of internal hollow regions through the Kirkendall effect [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e], and these regions can potentially operate as plasmonic hotspots; and iv) molecules have an opportunity to penetrate into bimetallic layers during the GR process [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. An additional advantage of Au@Ag alloys is that they can be formed \u003cem\u003evia\u003c/em\u003e GR at room temperature without additional heating or catalysts, providing excellent reproducibility. Therefore, comprehensive studies on the characteristics and applications of interior hotspots in Au@Ag bimetallic platforms are required.\u003c/p\u003e \u003cp\u003eIn the present study, we developed an Au@Ag material with interior hotspots by galvanically replacing the Ag in Ag-coated cotton fabric nanopillars with Au (Au@Ag/CFNPs), and applied the resulting platform to the label-free detection of two plasticizers (\u003cem\u003ei.e.\u003c/em\u003e, BBP and BPA). The supporting nanostructures were directly defined on in-vitro diagnostic (IVD) cotton fabric (CF) through a maskless plasma etching process. The sacrificial Ag layer was rendered on the CFNPs (Ag/CFNPs) using the mirror reaction (MR), and then the Au@Ag bimetal was synthesized \u003cem\u003evia\u003c/em\u003e the GR in the presence of analyte molecules. The presence and function of interior hotspots within the Au@Ag/CFNPs were experimentally and theoretically verified. The repeatability and sensitivity of the proposed platform were estimated for individual plasticizers, and these reference data were used to evaluate the sensing performance of the platform when applied to an actual plastic sample. A lateral flow assay (LFA) kit templated with an Ag/CFNP test line was designed for convenient detection of plasticizers.\u003c/p\u003e"},{"header":"2. Experimental section","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1. Materials\u003c/h2\u003e \u003cp\u003eSilver nitrate (AgNO\u003csub\u003e3\u003c/sub\u003e), potassium hydroxide (KOH), D-(+)-glucose, gold chloride trihydrate (HAuCl\u003csub\u003e4\u003c/sub\u003e\u0026middot;3H\u003csub\u003e2\u003c/sub\u003eO), methylene blue (MB), BBP, and BPA were purchased from Sigma-Aldrich (St. Louis, MO, USA). Ammonia solution (NH\u003csub\u003e3\u003c/sub\u003e, 28%) was purchased from Junsei (Tokyo, Japan). Anhydrous ethyl alcohol (99.9%) was purchased from Samchun (Gangnam, South Korea). Sample pads (319 grade) consisting of CFs were obtained from Ahlstrom (Helsinki, Finland).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2. Fabrication of the Au@Ag/CFNP platform\u003c/h2\u003e \u003cp\u003eThe supporting CFNPs were fabricated using a maskless plasma etching process. CF pads were loaded into a reactive-ion etching chamber, and then the base pressure was set to 7.2 mTorr. Under an O\u003csub\u003e2\u003c/sub\u003e flow rate of 52 sccm and a radio-frequency power of 100 W (13.56 MHz), O\u003csub\u003e2\u003c/sub\u003e plasma treatment was applied to the substrates for 2 min. Au@Ag/CFNPs were prepared \u003cem\u003evia\u003c/em\u003e a two-step chemical process: Ag MR followed by GR. First, Tollens\u0026rsquo; reagent containing 0.5 M AgNO\u003csub\u003e3\u003c/sub\u003e (5 mL), 0.8 M KOH (660 \u0026micro;L), and NH\u003csub\u003e3\u003c/sub\u003e (680 \u0026micro;L) was applied to the CFNPs, followed by injection of a 0.5 M glucose solution (660 \u0026micro;L). A sacrificial Ag layer formed on the CFNPs during the 10-min MR process. The resulting Ag/CFNP pads were rinsed with de-ionized water and then dried under ambient conditions. The as-prepared pads were cut into 5 mm \u0026times; 5 mm squares. To generate interior hotspots, an \u003cem\u003ein-situ\u003c/em\u003e GR process was applied by adding a 0.5 mM HAuCl\u003csub\u003e4\u003c/sub\u003e solution to the Ag/CFNPs in the presence of the solution to be tested.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3. SERS measurements\u003c/h2\u003e \u003cp\u003eAn Ocean Optics portable probe spectrometer system (UQEPRO-Raman) was used to record real-time SERS spectra during the GR process. The samples were irradiated with a 785 nm laser with an optical power of 40 mW in a normal direction. SERS signals were collected for MB and plasticizers using acquisition times of 10 s and 30 s, respectively.\u003c/p\u003e \u003cp\u003e \u003cem\u003e2.4. LFA-SERS assays\u003c/em\u003e \u003c/p\u003e \u003cp\u003eTo construct the LFA-SERS kit, a sample pad of dimensions 4.5 mm \u0026times; 500 mm was prepared. The Ag/CFNPs were fabricated with an area of 4.5 mm \u0026times; 30 mm to be used as a test line. The as-prepared pads were mounted on an adhesive backing membrane. A mixture (100 \u0026micro;L) of 0.5 mM HAuCl4 and test solution with a ratio of 1:1 was injected into the kit. After incubation for 2 min, SERS signals were measured from the plasmonic test line.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.5. Theoretical computations\u003c/h2\u003e \u003cp\u003eThe \u003cem\u003eE\u003c/em\u003e-field profiles of the Ag/CFNP (\u003cem\u003ei.e.\u003c/em\u003e, GR time of 0 s) and Au@Ag/CFNP (\u003cem\u003ei.e.\u003c/em\u003e, GR time of 60 s) platforms were calculated numerically with the finite-difference time domain (FDTD) method using a commercial program (Ansys Lumerical 2021 R1.2). The structural parameters were extracted from scanning transmission electron microscopy (STEM) analyses. For the Ag/CFNPs, a conical CFNP (radius 90.9 nm, height 285.7 nm) was placed in a cuboid Ag layer (372.6 nm \u0026times; 372.6 nm \u0026times; 285.7 nm). The Ag head (radius 117.8 nm, height 90.9 nm) was located at the top of the CFNP. For the Au@Ag/CFNPs, void areas were constructed between the CFNP and Au@Ag shell. The bimetallic nanoporous layer was generated by mixing Au and Ag nanoparticles in a ratio of 1:9, and was applied to the cuboid layer (372.6 nm \u0026times; 372.6 nm \u0026times; 285.7 nm) and shell (radius 126.3 nm, height 109.0 nm). Assuming highly symmetric nanostructures, periodic boundary conditions were applied to the \u003cem\u003ex\u003c/em\u003e- and \u003cem\u003ey\u003c/em\u003e-axes, and a perfectly matched layer was used at the top and bottom layers. An \u003cem\u003ex\u003c/em\u003e-polarized plane wave (785 nm) was normally incident on the designed structures. The mesh scale was fixed at 0.5 nm. For the optical parameters at 785 nm, the dielectric constants of Au and Ag were used, specifically \u003cem\u003eε\u003c/em\u003e\u003csub\u003eAu\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;\u0026minus;\u0026thinsp;21.64\u0026thinsp;+\u0026thinsp;\u003cem\u003ei\u003c/em\u003e0.74 and \u003cem\u003eε\u003c/em\u003e\u003csub\u003eAg\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;\u0026minus;\u0026thinsp;30.87\u0026thinsp;+\u0026thinsp;\u003cem\u003ei\u003c/em\u003e2.99, respectively. The refractive indices of the CF pad and background water were set to 1.55 and 1.33, respectively.\u003c/p\u003e \u003cp\u003e \u003cem\u003e2.6. Characterizations\u003c/em\u003e \u003c/p\u003e \u003cp\u003eThe morphological properties of the Ag/CFNPs and Au@Ag/CFNPs were observed by field-emission scanning electron microscopy (FE-SEM; JSM-6700F, Jeol, Tokyo, Japan) and STEM (JEM-ARM200F, Jeol, Tokyo, Japan).\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results and discussion","content":"\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e3.1. Strategy of the Au@Ag/CFNPs for detection of plasticizers\u003c/h2\u003e \u003cp\u003eThe characteristics of plasmonic hotspots (\u003cem\u003ei.e.\u003c/em\u003e, scale, density, and occupancy rate) directly influence the SERS detection performance. A higher hotspot density increases the probability of molecules being located in narrower plasmonic active regions, making nanoporous platforms desirable for SERS applications [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. In this study, the GR process coupled with the Kirkendall effect was used to create hollow regions in bimetallic nanostructures. The fabrication procedure of the proposed platform is shown in Scheme \u003cspan refid=\"Sch1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. For practical applications, the sample pad, an IVD CF, was selected as a base substrate. To define the supporting CFNPs, O\u003csub\u003e2\u003c/sub\u003e plasma was applied to the pads (Scheme \u003cspan refid=\"Sch1\" class=\"InternalRef\"\u003e1\u003c/span\u003e(a)). After reaction with oxygen plasma, polar dangling bonds (resulting from chain breakage) and hydroxyl groups became the predominant active sites onto which Ag ions were adsorbed for seed formation [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. An electroless MR process was employed to deposit the sacrificial Ag layer on the CFNPs (Scheme \u003cspan refid=\"Sch1\" class=\"InternalRef\"\u003e1\u003c/span\u003e(b)). The Ag layer was synthesized based on the redox process between Tollens\u0026rsquo; reagent and glucose according to the equation:\u003c/p\u003e \u003cp\u003e \u003cdiv id=\"Equ1\" class=\"Equation\"\u003e \u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equ1\" name=\"EquationSource\"\u003e\n$${2\\left[{\\text{A}\\text{g}\\left({\\text{N}\\text{H}}_{3}\\right)}_{2}\\right]}^{+}+{\\text{C}}_{5}{\\text{H}}_{11}{\\text{O}}_{5}\\text{C}\\text{H}\\text{O}+{2\\text{O}\\text{H}}^{-}\\to2\\text{A}\\text{g}+{\\text{C}}_{5}{\\text{H}}_{11}{\\text{O}}_{5}\\text{C}\\text{O}\\text{O}\\text{H}+{\\text{H}}_{2}\\text{O}+4{\\text{N}\\text{H}}_{3}$$\u003c/div\u003e \u003cdiv class=\"EquationNumber\"\u003e1\u003c/div\u003e\u003c/div\u003e.\u003c/p\u003e \u003cp\u003eNext, the GR process proceeded by injecting a HAuCl\u003csub\u003e4\u003c/sub\u003e solution onto the Ag/CFNPs (Scheme \u003cspan refid=\"Sch1\" class=\"InternalRef\"\u003e1\u003c/span\u003e(c)). The GR is a spontaneous reaction between two or more noble metals driven by differences in \u003cem\u003eE\u003c/em\u003e\u003csup\u003eo\u003c/sup\u003e. The standard \u003cem\u003eE\u003c/em\u003e\u003csup\u003eo\u003c/sup\u003e values of AuCl\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e/Au and Ag\u003csup\u003e+\u003c/sup\u003e/Ag are approximately 1.5 V and 0.8 V versus the standard hydrogen electrode, respectively [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. Because the material with higher standard \u003cem\u003eE\u003c/em\u003e\u003csup\u003eo\u003c/sup\u003e accepts electrons (\u003cem\u003ei.e.\u003c/em\u003e, reduction) from the one with lower standard \u003cem\u003eE\u003c/em\u003e\u003csup\u003eo\u003c/sup\u003e (\u003cem\u003ei.e.\u003c/em\u003e, oxidation), the Au@Ag bimetal is constructed by the following stoichiometric equation:\u003cdiv id=\"Equ2\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equ2\" name=\"EquationSource\"\u003e\n$${3\\text{A}\\text{g}}_{\\left(s\\right)}+{{{\\text{A}\\text{u}\\text{C}\\text{l}}_{4}}^{-}}_{\\left(aq\\right)}\\to3{{\\text{A}\\text{g}}^{+}}_{\\left(aq\\right)}+{\\text{A}\\text{u}}_{\\left(s\\right)}+{4{\\text{C}\\text{l}}^{-}}_{\\left(aq\\right)}$$\u003c/div\u003e\u003cdiv class=\"EquationNumber\"\u003e2\u003c/div\u003e\u003c/div\u003e.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eThe diffusion rates of Au (\u003cem\u003ei.e.\u003c/em\u003e, inward direction) and Ag (\u003cem\u003ei.e.\u003c/em\u003e, outward direction) are not balanced because of the atomic exchange ratio of Au:Ag\u0026thinsp;=\u0026thinsp;1:3 (\u003cem\u003ei.e.\u003c/em\u003e, the Kirkendall effect), and this imbalance contributes to the creation of hollow regions in the sacrificial layer. Such spaces play a dual role as plasmonic interior hotspots and molecular diffusion pathways. During the \u003cem\u003ein-situ\u003c/em\u003e GR process, in which the test solution is present during hotspot formation, analyte molecules can be embedded in the hollow regions, possibly leading to a massive enhancement of their SERS signals. The proposed LFA-SERS kit, including a plasmonic membrane (herein, the IVD sample pad), exploits this signal amplification to enable the convenient detection of toxic and hazardous substances.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e3.2. Morphological and optical characteristics of the Au@Ag/CFNPs via the GR process\u003c/h2\u003e \u003cp\u003eDuring Au@Ag/CFNP formation \u003cem\u003evia\u003c/em\u003e the GR process, the following phenomena are typically observed: i) the creation of hollow regions due to the Kirkendall effect, ii) conversion of Ag to Au@Ag bimetal, and iii) re-reduction of a portion of oxidized Ag ions [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. Therefore, the entire structural domain rapidly increases with processing time due to the formation of outer bimetallic layers [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. This behavior was confirmed by examining the morphological changes of the Au@Ag/CFNPs at different GR times. Herein, the Ag/CFNPs were prepared by applying the MR (0.5 M AgNO\u003csub\u003e3\u003c/sub\u003e solution for 10 min) to the CFNPs (O\u003csub\u003e2\u003c/sub\u003e plasma treatment for 2 min), leading to the formation of an uniformly distributed Ag layer (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e(a)). At a GR time of 30 s, partial Ag dissociation was observed (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e(b)). For longer GR times, bimetallic layers containing a large number of hollow sites grew, and eventually covered the underlying structures (Figs.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e(c)\u0026ndash;(d)).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eOur previous research established that the signal enhancement in EC-SERS relies on the coupling of interior hotspots with environmental hotspots [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. Therefore, we optimized the supporting Ag/CFNP platform to achieve maximum efficiency. Under plasma treatment, the polymeric nanostructures are generated by the following surface dynamics: selective etching, surface migration, agglomeration, and coalescence [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. In this work, the CFNPs became agglomerated 3 min after the application of O\u003csub\u003e2\u003c/sub\u003e plasma to the CF substrate (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). Given the morphological profile of the CFNPs (\u003cem\u003ei.e.\u003c/em\u003e, density and aspect ratio), the optimum O\u003csub\u003e2\u003c/sub\u003e plasma treatment time was determined to be 2 min. Next, the influence of MR parameters (\u003cem\u003ei.e.\u003c/em\u003e, synthesis time and AgNO\u003csub\u003e3\u003c/sub\u003e concentration) on the optical signal enhancement was investigated. To evaluate the SERS activities of the Au@Ag/CFNPs, herein, MB Raman dyes were used. During the MR process with 0.5 M AgNO\u003csub\u003e3\u003c/sub\u003e, Ag seeds were formed during the initial stage and then grew with synthesis time (Figs. S2(a)\u0026minus;(d)). The most intense MB signal was achieved from the Ag/CFNPs synthesized using an MR time of 10 min (Figs. S2(e)\u0026minus;(f)). At earlier times (\u003cem\u003ei.e.\u003c/em\u003e, 1 and 5 min), the plasmonic resonance was decreased due to the lower density of Ag nanostructures, while at later stages (\u003cem\u003ei.e.\u003c/em\u003e, longer than 10 min), the plasmonic resonance was degraded by significant structural inhomogeneity (\u003cem\u003ei.e.\u003c/em\u003e, non-uniformity). Experiments on Ag/CFNPs generated using different AgNO\u003csub\u003e3\u003c/sub\u003e concentrations showed that the optimum concentration was 0.5 M (Fig. S3).\u003c/p\u003e \u003cp\u003eTo optimize the GR process, experiments were performed in which the HAuCl\u003csub\u003e4\u003c/sub\u003e concentration (\u003cem\u003ei.e.\u003c/em\u003e, replacement ratio) was varied. The SERS signals showed the highest intensity at a Au precursor concentration of 0.5 mM (Fig. S4). Using the optimum conditions, the time evolution of the SERS signal at 1612 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e during the GR process in which Au@Ag/CFNPs were synthesized in the presence of MB (MB\u0026thinsp;\u0026minus;\u0026thinsp;Au@Ag/CFNPs) was measured (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e(e)). The peak intensity reached saturation 40 s after injection of the Au precursor. To ensure reliable analysis, the optimum GR time was set to 60 s. To verify the superior function of interior hotspots, SERS spectra collected from three systems were compared: Ag/CFNPs to which MB was added (MB\u0026ndash;Ag/CFNPs), \u003cem\u003ein-situ\u003c/em\u003e MB\u0026ndash;Au@Ag/CFNPs, and Au@Ag/CFNPs to which MB was added after Au@Ag/CFNP synthesis (\u003cem\u003ei.e.\u003c/em\u003e, post-addition) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e(f)). The spectrum obtained from the MB\u0026thinsp;\u0026minus;\u0026thinsp;Ag/CFNPs showed negligible SERS signals (\u003cem\u003eI\u003c/em\u003e\u0026thinsp;~\u0026thinsp;20) because the MB molecules moving \u003cem\u003evia\u003c/em\u003e Brownian motion were barely adsorbed onto the environmental hotspots (\u003cem\u003ei.e.\u003c/em\u003e, a low occupancy rate). When the dye was added to the already formed Au@Ag/CFNPs, SERS peaks were observed but with low intensity (\u003cem\u003eI\u003c/em\u003e\u0026thinsp;~\u0026thinsp;1.8k). Although both interior and environmental hotspots were present in this case, delivery of MB molecules into the narrow interstitials was difficult due to the high surface tension of the solvent. In the case of the MB\u0026ndash;Au@Ag/CFNPs formed during \u003cem\u003ein-situ\u003c/em\u003e GR, by contrast, the dyes had the opportunity to penetrate into the desired areas during the dynamic atomic exchange that occurs in the GR, resulting in a remarkable amplification of the optical signals (\u003cem\u003eI\u003c/em\u003e\u0026thinsp;~\u0026thinsp;60.3k). Accordingly, the SERS intensity for the \u003cem\u003ein-situ\u003c/em\u003e MB\u0026thinsp;\u0026minus;\u0026thinsp;Au@Ag/CFNPs was approximately 33.5-fold higher than that of the post-addition of MB to Au@Ag/CFNPs. These results indicate that not only the plasmonic performance but also the molecular distribution play a crucial role in determining sensing efficiency of SERS platforms.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e3.3. Investigation of the GR-induced interior hotspots in Au@Ag/CFNPs\u003c/h2\u003e \u003cp\u003eTo observe the generation of the internal hollow regions in the Au@Ag/CFNPs, STEM analyses were conducted on the Ag/CFNPs and the Au@Ag/CFNPs formed after a GR time of 60 s (Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e(a)\u0026minus;(b)). The STEM image of the Ag/CFNPs shows a solid Ag layer on the supporting CFNPs. In this system, environmental hotspots are only provided by the transverse mode between neighboring Ag heads. The STEM image of the Au@Ag/CFNPs, by contrast, shows hollow regions with dimensions of 6\u0026ndash;11 nm within the bimetallic layer, where the formation of the hollow regions is attributed to the Kirkendall effect. To confirm the formation of a bimetallic layer, elemental mapping analyses were performed (Fig. S5). After the GR process, the pure Ag component was transformed to Au@Ag alloys with an atomic percentage ratio of ~\u0026thinsp;9.7:90.3. Au and Ag are miscible because they belong to the same space group (\u003cem\u003ei.e.\u003c/em\u003e, cubic \u003cem\u003eFm̅\u003c/em\u003e3\u003cem\u003em\u003c/em\u003e) and their lattice constants are similar (Au: 4.079 \u0026Aring; and Ag: 4.086 \u0026Aring;) [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, 38]. In the high-resolution TEM image (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e(c)), lattice fringes with a \u003cem\u003ed\u003c/em\u003e-spacing of 0.24 nm, corresponding to the (111) planes of Au and Ag, are observed, demonstrating the highly crystalline nature of Au@Ag/CFNPs [29]. In addition, selected area electron diffraction analysis showed patterns consistent with a face-centered cubic structure [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e, 40], as shown in the inset of Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e(c).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo theoretically verify the activation of interior hotspots, the \u003cem\u003eE\u003c/em\u003e-field profiles of Ag/CFNPs and Au@Ag/CFNPs formed after a GR time of 60 s were calculated using the FDTD method (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e(d)). The simulation models were constructed using the geometric parameters extracted from the STEM images. The following assumptions were made to simplify the structures: i) the Ag/CFNPs are periodically arranged, ii) the Ag layers are filled in the space between the CFNPs, iii) the Ag heads are hemispheres, and iv) the bimetallic layer is comprised of a 1:9 mixture of Au and Ag nanoparticles according to the elemental mapping result. For the Ag/CFNPs, weak \u003cem\u003eE\u003c/em\u003e-fields were produced between the Ag heads because the heads are separated by a large gap and the strength of \u003cem\u003eE\u003c/em\u003e-fields rapidly diminishes with gap distance (\u003cem\u003ei.e.\u003c/em\u003e, LSPR). After the GR, the field intensity became stronger because i) bimetallic layers containing internal hollow regions were generated and ii) the growth of the entire structural domain reduced the gap between the Au@Ag heads. Resonance coupling between the interior and environmental hotspots may further enhance molecular signals. According to the fourth power approximation, the theoretical enhancement factor of the Au@Ag/CFNPs was estimated to be 3.7 \u0026times; 10\u003csup\u003e7\u003c/sup\u003e, which was much greater than that of the Ag/CFNPs (1.2 \u0026times; 10\u003csup\u003e3\u003c/sup\u003e). These results may explain the differences in spectral enhancement observed among the structural models in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e(f).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e3.4. SERS activities of in-situ MB\u0026thinsp;\u0026minus;\u0026thinsp;Au@Ag/CFNPs\u003c/h2\u003e \u003cp\u003eThe SERS performance (\u003cem\u003ei.e.\u003c/em\u003e, repeatability and sensitivity) of the Au@Ag/CFNPs was investigated to assess the suitability of the platform for reliable molecular assays in practical settings. Using the optimum protocol, the SERS spectra of the Au@Ag/CFNPs formed in the presence of 100 nM MB dye solutions were collected from 20 different chips (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e(a)). The SERS spectra from the 20 chips showed high consistency, and each spectrum showed features characteristic of MB. Herein, the MB peaks at 445, 772, 1390, and 1612 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e were selected for further analyses; the peak assignments of MB are summarized in Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. To calculate the relative standard deviation (RSD), the intensity profiles at the analytic peaks were extracted (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e(b)). The RSD values were 6.46%, 9.14%, 7.13%, and 7.00% at 445, 772, 1390, and 1612 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, respectively, indicating excellent signal uniformity. The excellent repeatability of the method can be attributed to i) the formation of plasmonic hotspots with high density and ii) the GR process being spontaneous and stable.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFor quantitative analyses, the signals from the Au@Ag/CFNPs formed in the presence of MB solutions with different concentrations (\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;5) were measured (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e(c)). Characteristic peaks of MB were observed in all of the spectra, and their intensities gradually diminished with decreasing concentration because of the reduction of the molecular adsorption probability described by the Langmuir model [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. The proposed platform showed femtomolar sensitivity, demonstrating the superior plasmonic activities of the interior hotspots in the Au@Ag bimetals. To investigate the quantitative correlation between peak intensity and MB concentration, the intensity (\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;5) of each analytic peak was plotted against concentration on a log-log scale (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e(d)). For each peak, a highly linear relationship was observed with a correlation coefficient (R\u003csup\u003e2\u003c/sup\u003e) of 0.99. The limit-of-detection (LOD) was also calculated using the equation\u003cdiv id=\"Equ3\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equ3\" name=\"EquationSource\"\u003e\n$$\\text{L}\\text{O}\\text{D}=\\frac{3\\times{S}_{\\text{b}}}{m}$$\u003c/div\u003e\u003cdiv class=\"EquationNumber\"\u003e3\u003c/div\u003e\u003c/div\u003e,\u003c/p\u003e \u003cp\u003ewhere \u003cem\u003eS\u003c/em\u003e\u003csub\u003eb\u003c/sub\u003e is the standard deviation of blank measurements (\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;5) at the analytic peak and \u003cem\u003em\u003c/em\u003e is the slope of the calibration curve. The average LOD value for the \u003cem\u003ein-situ\u003c/em\u003e MB\u0026ndash;Au@Ag/CFNPs was estimated to be 184.2 aM. As a result, the proposed Au@Ag/CFNP platform, which has the advantages of narrow interstitials, large molecular participation, and stable reaction, is a promising technology for tracing concentrations of hazardous substances.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e3.5. Sensitivity evaluation of in-situ plasticizer\u0026thinsp;\u0026minus;\u0026thinsp;Au@Ag/CFNPs\u003c/h2\u003e \u003cp\u003eAccording to the RoHS 2 directive and REACH regulation, plasticizer concentrations must not exceed 10\u003csup\u003e3\u003c/sup\u003e ppm in manufactured plastic products. Therefore, detection systems must have a sensitivity below this level to enable reliable analysis of actual samples. In this work, the BBP and BPA were chosen as the analytic substances owing to their widespread usage in the manufacturing of plastics and epoxy resins [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Before the sensitivity evaluation, the activation of interior hotspots containing embedded plasticizers was investigated by comparing the SERS spectra of plasticizer\u0026thinsp;\u0026minus;\u0026thinsp;Ag/CFNP and \u003cem\u003ein-situ\u003c/em\u003e plasticizer\u0026thinsp;\u0026minus;\u0026thinsp;Au@Ag/CFNP systems (Fig. S6), where the plasticizer concentration was set to 5000 ppm. For both plasticizers, negligible SERS signals were obtained from the Ag/CFNPs, whereas their signals were clearly observed after the \u003cem\u003ein-situ\u003c/em\u003e GR process was applied. These results indicated that the lipophilic plasticizer molecules were able to penetrate into the narrow hollow regions effectively.\u003c/p\u003e \u003cp\u003eThe sensitivity of the proposed platform with GR-induced interior hotspots was evaluated for the detection of BBP and BPA. For the BBP\u0026ndash;Au@Ag/CFNP system (\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;5), representative SERS peaks at 646, 997, 1033, 1598, and 1725 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e were observed in the concentration range from 5 \u0026times; 10\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e to 5 \u0026times; 10\u003csup\u003e4\u003c/sup\u003e ppm (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e(a)). The peak assignments of BBP, consisting of an aryl alkyl ester of phthalic acid, are presented in detail in Table S2 [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e, \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. Among them, the peaks at 997 and 1033 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e were selected as analytic criteria; these peaks correspond to C\u0026thinsp;\u0026minus;\u0026thinsp;C in-plane bending in the secondary benzene ring and aromatic ring breathing in the primary benzene ring of BBP. From the logarithmic calibration curve, the R\u003csup\u003e2\u003c/sup\u003e and average LOD values were calculated to be \u0026ge;\u0026thinsp;0.97 and 15.6 ppb, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e(b)). In the case of BPA (\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;5), the spectral characteristic features of the compound were detectable down to a concentration of 0.5 ppm (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e(c)). The peak assignments of BPA, which contains a diphenylmethane bearing two hydroxyphenyl groups in the \u003cem\u003epara\u003c/em\u003e positions, are summarized in Table S3 [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e, \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. Plots of the intensities at 642 and 877 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e versus BPA concentration reveal a highly linear correlation, with R\u003csup\u003e2\u003c/sup\u003e values of \u0026ge;\u0026thinsp;0.95 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e(d)). Based on the three standard deviation equation, the average LOD was found to be 219.7 ppb. These results indicate that the proposed platform is a viable alternative to current standard techniques (\u003cem\u003ee.g.\u003c/em\u003e, pyrolysis, GC\u0026ndash;MS, and HPLC) for practical assays.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e3.6. Detection of actual samples based on the in-situ Au@Ag/CFNP platform\u003c/h2\u003e \u003cp\u003eTo demonstrate the practical applicability of the proposed platform, the system was applied to plasticizer detection in a polycarbonate (PC) plastic product. As reported in our previous study [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e], the following protocol was employed to obtain the test solution: i) the PC product was cut into small pieces, ii) 0.3 g of the sample was immersed in ethanol, iii) the solution was left at room temperature for 2 h to extract plasticizers, and iv) the filtration was applied to remove plastic products [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. During the extraction, no heating or pressure was applied. From the HPLC analysis, the plasticizer in the leaching solution was found to BPA with a concentration of ~\u0026thinsp;10 ppm. The SERS spectrum of the test sample matched the representative spectrum of BPA with an excellent signal-to-noise ratio (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e(a)). This was attributed to i) negligible background noise originating from the Au@Ag/CFNPs, ii) the boosting of optical signals by the GR-induced interior hotspots, and iii) the diffusion of unwanted large molecules to the bottom area of the CF substrate \u003cem\u003evia\u003c/em\u003e pores. Because the plastic extraction process generates a leaching solution containing other compounds in addition to BPA, the BPA molecules must compete with these other compounds for access to the plasmonic hotspots. As a result, the spectrum of the leaching solution exhibited slightly lower intensity than the reference spectrum (\u003cem\u003ei.e.\u003c/em\u003e, 10 ppm BPA dissolved in ethanol). For example, the intensity at 877 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e of the leaching solution was close to the fitting line of the quantitative reference data (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e(b)). Therefore, the proposed platform demonstrated great potential for detecting very low levels of toxic substances leached from plastic products, which is important for early screening in industrial applications.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e3.7. LFA application\u003c/h2\u003e \u003cp\u003eFor the on-site detection of plasticizers, convenient, simple, and cost-effective plasmonic sensing chips are required. For this purpose, a prototype LFA-SERS kit based on a Ag/CFNP strip was prepared (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e(a)). The assembly of the kit was facilitated by the formation of plasmonic nanostructures on the IVD material (\u003cem\u003ei.e.\u003c/em\u003e, CFs). The LFA kit was designed with three compartments: i) a sample pad consisting of pristine CFs, ii) a Ag/CFNP strip of length 3 cm as an optical test line, and iii) an absorption pad. In the kit, the sample pad served as a filter to prevent large interferents (\u003cem\u003ee.g.\u003c/em\u003e, microplastics) reaching the test line. For sample loading, the plasticizer solution was mixed with a 0.5 mM HAuCl\u003csub\u003e4\u003c/sub\u003e solution at a volume ratio of 1:1, and a 100 \u0026micro;L droplet was applied to the sample pad. The mixture solution was driven by the capillary force. To ensure the formation of interior hotspots, the measurement was conducted 2 min after sample loading.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe prototype LFA-SERS kit was tested using solutions with various BBP concentrations. Spectra collected from the kit (\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;3) including the Au@Ag/CFNPs embedded with BBP are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e(b). The BBP molecules were successfully detected in the concentration range of 10\u003csup\u003e1\u003c/sup\u003e\u0026minus;10\u003csup\u003e4\u003c/sup\u003e ppm. Notably, the loaded samples covered both the internal and external surfaces, leading to initiation of the GR. For comparison with reference data, the intensity profiles at 997 and 1033 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e are displayed in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e(c). The intensity levels in the LFA-SERS spectrum are lower than those in the reference spectrum because not all of the target molecules and Au precursors were involved in the formation of interior hotspots (\u003cem\u003ei.e.\u003c/em\u003e, component loss). From the logarithmic calibration, the R\u003csup\u003e2\u003c/sup\u003e and average LOD values were found to be ~\u0026thinsp;0.90 and 1.4 ppm, respectively. Although the LFA-SERS kit showed lower detection performance compared with the reference data, it remains a rapid, sensitive, and high-throughput diagnostic tool for the early screening of plasticizers in plastic products.\u003c/p\u003e \u003c/div\u003e"},{"header":"4. Conclusions","content":"\u003cp\u003eWe reported the development of bimetallic nanostructures templated with interior hotspots on an IVD material (\u003cem\u003ei.e.\u003c/em\u003e, Au@Ag/CFNPs) for the ultrasensitive and on-site SERS detection of plasticizers. The plasmonic materials were prepared \u003cem\u003evia\u003c/em\u003e a two-step chemical synthesis: Ag MR followed by GR. The internal hollow regions (\u003cem\u003ee.g.\u003c/em\u003e, interstitials, cracks, and voids) induced by the Kirkendall effect played a critical role as plasmonic hotspots and molecular pathways when the target analyte molecules were present during the process (\u003cem\u003ei.e.\u003c/em\u003e, \u003cem\u003ein-situ\u003c/em\u003e GR). The activation of such dual-function interior hotspots was confirmed by comparison with structural models in which the target analyte molecules were added after the GR had completed. The enhancement factor of Au@Ag/CFNPs was calculated by FDTD simulation to be 3.7 × 10\u003csup\u003e7\u003c/sup\u003e. For MB dye, the Au@Ag/CFNP chips showed reliable sensing operation (\u003cem\u003ei.e.\u003c/em\u003e, RSDs \u0026lt;10%) with picomolar sensitivity. The proposed platform also detected the lipophilic plasticizers BBP and BPA at sub-ppm concentrations. The SERS spectrum of the leaching solution derived from a PC plastic product showed features characteristic of BPA without significant background noise. For convenient on-site diagnosis, an LFA kit templated with Ag/CFNPs was designed. Despite partial loss of the analytes and Au precursor during capillary flow into the sample pad, the LFA-SERS kit successfully traced the BBP in the concentration range of 10\u003csup\u003e1\u003c/sup\u003e−10\u003csup\u003e4\u003c/sup\u003e ppm. Therefore, the Au@Ag/CFNP platform with dual-function interior hotspots has great potential for detecting ecotoxicological substances in an ultrasensitive, rapid, and reliable manner, making it a promising technology for use in industrial applications.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eDeclaration of Competing Interes\u003c/strong\u003e\u003cstrong\u003ets\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare\u0026nbsp;that\u0026nbsp;they\u0026nbsp;have\u0026nbsp;no\u0026nbsp;known\u0026nbsp;competing\u0026nbsp;financial\u0026nbsp;interests\u0026nbsp;or\u0026nbsp;personal\u0026nbsp;relationships\u0026nbsp;that\u0026nbsp;could\u0026nbsp;have\u0026nbsp;appeared\u0026nbsp;to\u0026nbsp;influence\u0026nbsp;the\u0026nbsp;work\u0026nbsp;reported\u0026nbsp;in\u0026nbsp;this\u0026nbsp;paper.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSupplementary Information\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe online version contains supplementary material available at\u003c/p\u003e\n\u003cp\u003eAcknowledgments\u003c/p\u003e\n\u003cp\u003eThis research was supported by the Fundamental Research Program (PNKB010) of the Korea Institute of Materials Science (KIMS), Republic of Korea.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSoo Hyun Lee: Analysis and interpretation of data,\u0026nbsp;Investigation,\u0026nbsp;Writing-Original draft preparation, Validation,\u0026nbsp;Visualization.\u0026nbsp;ChaeWon Mun:\u0026nbsp;Investigation,\u0026nbsp;Methodology. Jun-Yeong Yang:\u0026nbsp;Investigation.\u0026nbsp;Seunghun Lee: Investigation.\u0026nbsp;Sung-Gyu Park:\u0026nbsp;Conceptualization, Writing- Reviewing and Editing,\u0026nbsp;Supervision, Project administration, Funding acquisition.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data that support the findings of this study are available from the corresponding author upon reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eClinical Trial Number\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eClinical trial number: not applicable.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eLuo Q, Liu Z, Yin H, Dang Z, Wu P, Zhu N, et al. 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Nanomaterials. 2021;11:881. https://doi.org/10.3390/nano11040881.\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Scheme 1","content":"\u003cp\u003eScheme 1 is available in the Supplementary Files section.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"discover-nano","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"narl","sideBox":"Learn more about [Discover Nano](https://www.springer.com/journal/11671)","snPcode":"11671","submissionUrl":"https://submission.nature.com/new-submission/11671/3","title":"Discover Nano","twitterHandle":"@SpringerOpen","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Discover Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Surface-enhanced Raman spectroscopy, Galvanic reaction, Interior hotspots, Plasticizers, Lateral flow assay","lastPublishedDoi":"10.21203/rs.3.rs-9024229/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9024229/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe impacts of toxic environmental substances such as plasticizers on human health have been intensively studied in recent years. For ultrasensitive and reliable detection of plasticizers, in the present work, we developed a bimetallic surface-enhanced Raman spectroscopy (SERS) platform in which Ag in Ag-coated cotton fabric nanopillars were subjected to GR with Au (Au@Ag/CFNPs) in the presence of analyte molecules. The hollow regions, which originated from the imbalanced stoichiometric ratio between Au and Ag, functioned as the plasmonic interior hotspots and molecular pathways. The spontaneous galvanic reaction (GR), based on the intrinsic material properties (\u003cem\u003ei.e.\u003c/em\u003e, reduction potential), exhibited repeatable SERS signals with relative standard deviation values of \u0026lt;\u0026thinsp;10%. For the plasticizers such as benzyl butyl phthalate (BBP) and bisphenol A (BPA), the proposed platform demonstrated sub-ppm sensitivity and a linear relationship between signal intensity and analyte concentration. To investigate the feasibility of the proposed platform in practical applications, a test solution extracted from an actual plastic sample containing BPA was measured using the Au@Ag/CFNP platform. Based on an in-vitro diagnostic cotton fabric (CF) used as a base material, a lateral flow assay (LFA) kit templated with Ag/CFNPs was prepared. With the injection of BBP molecules and Au precursor, the SERS signals could be detected from the LFA-SERS kit down to a BBP concentration of 10 ppm. The results indicate that the Au@Ag/CFNP platform with GR-induced interior hotspots could serve as an alternative to existing standard techniques (\u003cem\u003ee.g.\u003c/em\u003e, pyrolysis, gas chromatography\u0026ndash;mass spectrometry) for on-site early screening of plasticizers in food, daily products, and environments.\u003c/p\u003e","manuscriptTitle":"Au@Ag alloy containing interior hotspots galvanically prepared on an in-vitro diagnostic material for ultrasensitive and rapid detection of plasticizers","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-04-14 09:33:57","doi":"10.21203/rs.3.rs-9024229/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"editorInvitedReview","content":"","date":"2026-05-18T06:15:54+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"311802637652764675356789010845595130866","date":"2026-05-14T08:37:23+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-04-22T11:33:56+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"224152675600751473505774942258562810911","date":"2026-04-21T11:03:23+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"234587059916624396104691854011824670178","date":"2026-04-07T17:22:03+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-04-07T14:18:53+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-03-13T06:06:50+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-03-13T06:06:45+00:00","index":"","fulltext":""},{"type":"submitted","content":"Discover Nano","date":"2026-03-03T23:42:56+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"discover-nano","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"narl","sideBox":"Learn more about [Discover Nano](https://www.springer.com/journal/11671)","snPcode":"11671","submissionUrl":"https://submission.nature.com/new-submission/11671/3","title":"Discover Nano","twitterHandle":"@SpringerOpen","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Discover Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"a276df52-0cab-4571-a39d-5ebc6ae147d5","owner":[],"postedDate":"April 14th, 2026","published":true,"recentEditorialEvents":[{"type":"editorInvitedReview","content":"","date":"2026-05-18T06:15:54+00:00","index":28,"fulltext":""},{"type":"reviewerAgreed","content":"311802637652764675356789010845595130866","date":"2026-05-14T08:37:23+00:00","index":27,"fulltext":""}],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-04-14T09:33:57+00:00","versionOfRecord":[],"versionCreatedAt":"2026-04-14 09:33:57","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9024229","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9024229","identity":"rs-9024229","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}