A review on recent advances in the applications of surface-enhanced Raman scattering in analytical chemistry

Journal Pre-proof A Review on Recent Advances in the Applications of Surface-Enhanced Raman Scattering in Analytical Chemistry Meikun Fan, Gustavo F.S. Andrade, Alexandre G. Brolo PII:

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Please cite this article as: M. Fan, G.F.S. Andrade, A.G. Brolo, A Review on Recent Advances in the Applications of Surface-Enhanced Raman Scattering in Analytical Chemistry, Analytica Chimica Acta, https://doi.org/10.1016/j.aca.2019.11.049. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier B.V.

A Review on Recent Advances in the Applications of SurfaceEnhanced Raman Scattering in Analytical Chemistry Meikun Fan†1, Gustavo F. S. Andrade†2, Alexandre G. Brolo*3,4

1

Faculty of Geosciences and Environmental Engineering, Southwest Jiaotong University,

Chengdu, Sichuan, 610031, China. 2

Centro de Estudos de Materiais, Departamento de Química, Instituto de Ciências Exatas,

Universidade Federal de Juiz de Fora, Campus Universitário s/n, CEP 36036-900, Juiz de Fora, Brazil. 3

Department of Chemistry, University of Victoria, PO Box 3055, Victoria, BC, Canada V8W

3V6. 4

Centre for Advanced Materials and Related Technology, University of Victoria, Canada, V8W

2Y2 †

These authors contributed the same.

Corresponding author: E-mail address: [email protected]

Keywords:

Metallic

nanostructures,

plasmonics,

SERS,

Raman

spectroscopy,

nanotechnology, nanoparticles

1

Abstract This review is focused on recent developments of surface-enhanced Raman scattering (SERS) applications in Analytical Chemistry. The work covers advances in the fabrication methods of SERS

substrates,

including

nanoparticles

immobilization

techniques

and

advanced

nanopatterning with metallic features. Recent insights in quantitative and sampling methods for SERS implementation and the development of new SERS-based approaches for both qualitative and quantitative analysis are discussed. The advent of methods for pre-concentration and new approaches for single-molecule SERS quantification, such as the digital SERS procedure, has provided additional improvements in the analytical figures-of-merit for analysis and assays based on SERS. The use of metal nanostructures as SERS detection elements integrated in devices, such as microfluidic systems and optical fibers, provided new tools for SERS applications that expand beyond the laboratory environment, bringing new opportunities for real-time field tests and process monitoring based on SERS. Finally, selected examples of SERS applications in analytical and bioanalytical chemistry are discussed. The breadth of this work reflects the vast diversity of subjects and approaches that are inherent to the SERS field. The state of the field indicates the potential for a variety of new SERS-based methods and technologies that can be routinely applied in analytical laboratories.

2

Summary List of abbreviations ....................................................................................................................... 4 Introduction ..................................................................................................................................... 7 1. Colloidal Systems and Assembles .............................................................................................. 9 1.1.

Progress in Nanoparticle synthesis ................................................................................. 11

1.2. Single-particle, dimers and controlled hotspots ................................................................. 12 1.3. Colloidal self-assembling and suspensions of large ordered structures ............................. 15 1.4. Self-assembly of NPs on planar and other types of platforms ........................................... 17 2. Fabricated Structures on solid supports .................................................................................... 20 2.1. Advanced nanofabrication methods ................................................................................... 20 2.2. Methods for large area fabrication ..................................................................................... 25 2.3. Low cost methods for plasmonic materials deposition over large area ............................. 28 3. Advances in SERS quantification ............................................................................................. 32 3.1. Quantification through large area mapping........................................................................ 34 3.2. Quantitative SERS near the single molecule regime ......................................................... 35 3.3. Other examples of quantitative SERS ................................................................................ 37 4. Advances in SERS substrates integrated to devices ................................................................. 38 4.1. Integration of SERS substrates on microfluidics devices .................................................. 38 4.2. Integration to optical fibers and microcapillaries based devices........................................ 43 5. Advances in Analytical Applications........................................................................................ 45 5.1. Concentrating solution phase samples with (super)hydrophobic and ominiphobic substrates ................................................................................................................................... 45 5.2. SERS substrate with direct sampling capability ................................................................ 46 6. Advances in Bioanalytical applications .................................................................................... 50 6.1. SERS Bioanalysis within cells ........................................................................................... 51 6.2. Bioassays for DNA and antigen/antibody recognition in body fluids ............................... 52 6.3. Analysis of nucleic acids by SERS .................................................................................... 56 6.4. Other bioanalytical applications ......................................................................................... 57 7. Other applications ..................................................................................................................... 60 Concluding Remarks ..................................................................................................................... 63 References ..................................................................................................................................... 64 3

List of abbreviations 1,4BDT - 1,4-benzenedithiol 3MPBA - 3-mercaptophenylboronic acid 4MBT - 4-methylbenzenethiol 4NBT - 4-nitrobenzenethiol Aβ40 - Amyloid-β40 AgNC - Ag nanocubes ALD – Atomic layer deposition APTMS – (3-Aminopropyl)trimethoxysilane BSA - bovine serum albumin CFU - colony forming units CTAB - cethyltrimethylammonium bromide CV - crystal violet CT - cholera toxin DMF – Dimethylformamide DSNB - 5,5'-dithiobis(succinimidyl-2-nitrobenzoate) DSP - dithiobis(succinimidyl propionate) EDTA - 2,2',2'',2'''-(Ethane-1,2-diyldinitrilo)tetraacetic acid ETPTA – Ethoxylated trimethylolpronanone triarylate GIANs - graphene-isolated-Au-nanocrystals GM1 - monosialoganglioside GSR - gunshot residues HCR – hybridization chain reaction

4

HPLC – High-performance liquid chromatography LoD – Limit-of-detection ManLAM - mannose-capped lipoarabinomannan NAAN - (N-(3-amidino)-aniline) NBA – Nile blue A NC – Nanocubes ND – Nanodimples NIL - nanoimprint lithography NT – Nanotubes p-PANI - emeraldine-salt polyaniline pATP – p-aminothiphenol PAH – Poly(allylamine hydrochloride) PCA - principal component analysis PIERS – Photo-induced enhanced Raman spectroscopy PDMS - Poly(dimethylsiloxane) PMMA – Poly(methyl 2-methylpropenoate) PMTTP - 4-(4-phenylmethanethiol)-2,2’: 6’,2’’-terpyridine PNAAN - poly(N-(3-amidino)-aniline) PNIPAM - poly-N-isopropylacrylamide PVA – Poly(vinyl alcohol) QS - quorum sensing R6G – Rhodamine-6G RFV - rapid vertical flow RIE – Reactive-ion etching RSD – Relative standard deviations 5

SACNT - super aligned carbon nanotubes SESORS - Spatially-offset SERS SIF – SERS intensity fluctuations SHIN – Shell-isolated nanoparticles Au SP - Gold superparticles ssDNA – single-stranded DNA TB - tuberculosis TLC – Thin-layer chromatography TSSEF – Total SERS substrate Enhancement Factor Zika-mAb - Zika monoclonal anti-NS1 antibodies

6

Introduction The Raman effect consists of inelastic light scattering by chemical species [1]. The energy of the scattered radiation carries vibrational information that is unique for a molecular system and the scattering intensity is proportional to the number density of molecules being probed. Therefore, the Raman scattering provides fundamental qualitative (molecular identification) and quantitative (concentration, for instance) information on molecular systems that are of uttermost importance to analytical chemists. However, normal Raman scattering is intrinsically weak, adding a significant limitation that precludes its wide application in chemical analysis. This means that although excellent quality Raman signatures can be obtained from many solids and pure liquids, the technique cannot be easily employed to study diluted solutions. This inherent limitation of normal Raman was overcome more than 40 years ago with the advent of surface-enhanced Raman scattering (SERS) [2, 3]. The SERS effect is an exceptional increase in Raman cross-section observed for molecules adsorbed on certain surfaces. Typically, nanostructured surfaces of gold, silver or copper are substrates that present efficient SERS response [4]. The efficiency of the SERS signal depends heavily on the geometrical characteristics of the metallic nanostructure [5], and a significant amount of the research in the field focus on the optimization of SERS substrates [6]. In fact, advances in nanofabrication, colloidal synthesis and self-assembly has led to hundreds of reports on different types of SERS substrates. Although the search for increased SERS efficiency is a main drive in this field, the transition of SERS into analytical applications requires a good level of reproducibility and repeatability. Historically, a reproducibility problem was identified earlier in SERS [7], due to the strong dependence of the SERS magnitude on the geometric characteristic of the metalmolecule system. Strong dependence here means that small variations in either molecular position or arrangement of the metal atoms within the nanostructure provoke very large fluctuations in SERS intensity. Those SERS intensity variations translate as large uncertainties in analytical calibration curves, large variability between measurements and overall unreliable determinations. It is a relative consensus (within and outside the field) that the reproducibility problem precludes the wide application of SERS in analytical chemistry. However, the current state-of-the-art of nanotechnology allows much better control over fabrication parameters of nanostructures, which has benefited SERS tremendously. A few years ago we co-authored a review on “SERS substrates and their application in analytical chemistry” suggesting that the 7

reproducibility problem is no longer a major obstacle for analytical applications of SERS [6]. There are actually several examples of SERS substrates in the literature that can provide large area spatial reproducibility and acceptable sample-to-sample variation in SERS intensities. Several commercial SERS substrates are now available in the market (together with miniaturized spectrometers), enabling a larger number of chemists to develop analytical methodologies based on SERS [8]. Significant breakthroughs in SERS substrate fabrication, quantification and application have been reported since our last review [6]. Our goal here is to highlight some of these advances and demonstrate that SERS is now more than ready to become a major detection and quantification tool in chemical analysis. SERS is a vibrant research area with a high level of activity. Therefore, before starting our account, it is also important to mention some of the most important reviews with relevance to analytical chemistry that has been published in the last few years. These recent review articles included detailed descriptions of advances in the chemical modification of metallic nanoparticles [9, 10], progress in synthetic methodologies to better control nanoparticles shape and size [11, 12] and new types of nanofabrication methods and tools [13-17]. The most explored area for SERS applications is by far biomedical and health research. The small footprint of SERS-based sensors is an ideal reason for their incorporation as a detection technology in biomedical and microfluidic devices [18, 19]. SERS methods were also developed to detect pathogens [20, 21] and to investigate biochemical processes in cells and tissues [22, 23]. The use of nanoparticles as diagnosis, treatment and drug delivery tools in nanomedicine indicates that SERS naturally fits as the detection mode of choice in those types of applications [24, 25]. The multiplexing potential of SERS probes has been now clearly demonstrated [26] and the exquisite sensitivity of the method allows both single particle [27, 28] and single molecule detection [29-32]. In this review, we will cover some of the main developments in SERS substrate synthesis, fabrication and application in analytical chemistry within the period between 2012 and 2019; i.e.; since our last review [6]. Most of the work that originated the field was discussed in [6], so, here we will focus only on the most recent progress. Considering the breadth of the SERS field, this account will not try to be comprehensive, but it will concentrate on new substrates, quantification and analytical and bio-analytical applications.

8

Before moving forward, a few words on the SERS enhancement factor may be useful to clarify the approach of the present review. The determination of SERS enhancement factor (SERS-EF) has been a standard procedure in reports of new substrates, as well as a metric for comparison between substrates. The now classical paper by Le Ru et al. report several procedures for the calculations of SERS-EF based on the surface characteristics (for instance, nanostructures either in suspension or immobilized on a fixed surface) [33]. It was, however, later shown that direct comparison of the SERS-EF from substrates that present different characteristics (colloidal suspensions (dynamic) vs. immobilized nanostructures (static), for instance) may be misleading [34]. It has also been widely recognized that a proper determination of SERS-EF require certain assumptions (for instance, the surface concentration of adsorbates) that can lead to large errors. Therefore, we decided not to report the SERS-EF in this review as a comparative metric. On the other hand, the SERS limit-of-detection (LoD) was used instead, when this type of data were available, to access the analytical significance of the method/substrate.

1. Colloidal systems and assemblies Colloidal systems are among the most studied platforms for SERS applications. The significant SERS efficiency of metallic nanoparticles (NPs), particularly Au, Ag and Cu, were early recognized and investigated by SERS researchers. The SERS characteristics of a colloidal system depend on the NPs shape, size and aggregation state. Colloidal aggregation leads to increased SERS, but if aggregation is performed without control, significant spatial, time and sample-to-sample variations might occur. Recent developments, summarized in Figure 1, have concentrated on different approaches for shape/size control, surface chemical modification and control over aggregation state and self-assembly. It is now possible to fine-tune the chemistry to allow quantitative determination of analytes in colloidal suspensions as well as on NPs immobilized on solid surfaces.

9

Figure 1. Selected examples of controlled shape, size distribution and aggregation state in colloidal systems. A) cucumber alike gold nanostars (NSt), adapted from ref. [28] © 2012 WileyVCH, used with permission; b) silica coated Au NSt, adapted from ref. [35]. © 2016 American Chemical Society; used with permission; c) Histogram of integrated SERRS intensities normalized by the average of individual nanoshells (N = 177)[36]. © 2011 American Chemical Society; used with permission; d) AuNPs dimerized by CB[n] with portal-to-portal separation rigidly fixed at 0.9 nm, adapted from ref. [37]. © 2011 American Chemical Society; used with permission; e) SERS peak intensity measured at 563 cm-1 and the product of extinction plotted as a function of the average aggregation number of the NR chains, adapted from ref. [38]. © 2011 American Chemical Society; used with permission; f) TEM images of the Au NPs film templated using supramolecular polymer, adapted from ref. [39]. © 2017 American Chemical Society; used with permission.

10

1.1. Progress in nanoparticle synthesis New types of technologies developed to achieve control over NPs shape and to impart surface modification have been reported. Spiked gold beads, nanostars (NSt) and core-shell particles have all been consolidated as excellent platforms for single particle SERS. For instance, spiked growth in high aspect ratio gold nanorods led to elongated “sea cucumber” beads, with sizes above 300 nm (Figure 1a) [28]. Single particle SERS were performed from this new type of Au NPs using a conventional Raman confocal microscope. The combination of self-assembled Au NSt onto fluorescently-labelled polystyrene beads was also introduced as a substrate for cell imaging [40]. The hybrid system yielded simultaneously fluorescence, SERS, and dark field imaging of cells. A new method for the synthesis of core-shell Au@Ag NPs was proposed [41]. It was found that protecting the Ag NPs with poly(styrenesulfonate) (PSS), instead of conventional poly(vinylpyrrolidone) (PVP), was a key step for Au shell rebuilding. The localized surface plasmon resonance (LSPR) of the Au@Ag system was tuned to the range between 470 and 800 nm, potentially enabling good quality SERS in the whole visible range. It is well-established that strong SERS occurs at hotspots located at metallic nanogaps. The control over these gaps in colloidal system is challenging, but the potential benefits in terms of SERS analysis from colloidal systems are tremendous. That justifies the large efforts towards tailored hotspots on single-particles in recent years. A particularly interesting protocol for the fabrication of nanogaps within single Au NPs involved an amphiphilic block copolymer selfassembled onto 20 nm Au NPs surfaces [42]. The block copolymer carried not only a Raman tag, but also redox active groups that assisted the in situ reduction of Au onto the copolymer-coated NPs. The benefit of the approach was that the thickness of the copolymer film on the Au NPs could be tailored into the sub-10 nm range. This nanogap control led to enormous SERS enhancement of the Raman tags. The authors used those Au NPs for the ultrasensitive labelling of cancer cells. Core/shell Si/metal nanostructures were also fabricated from high purity Si targets immersed in Au salt solutions [43]. An interesting method involving double-beam laser ablation in liquid was implemented to generate metal coated spherical Si NPs. The Si/Au NPs yielded a strong SERS signal capable of single monolayer detection. Several methodological advances had also improved the synthesis of silica/Au (or Ag) core-shell NPs [44]. Silica/metal nanoshells are 11

the most used plasmonic core/shell structures [45], but their synthesis used to be cumbersome and several steps required optimization. For instance, it was found that the nanoshell growth process (for both Ag and Ag) was highly dependent on the stirring rate. The optimum stirring rate that allowed control over the shell growth and resulted in smaller occurrence of external nucleation was found to be 190 rpm for Au and 1500 rpm for Ag. Overall, by optimizing the stirring and other steps in the procedure, the synthesis of Au or Ag nanoshells can now be accomplished within hours (instead of days and even weeks from earlier protocols). In addition, the batch-to-batch repeatability of the NPs was significantly improved. The resulting nanoshells have been proved to be a very effective SERS substrate. Au NSt is another class of NPs that gained a lot of prominence as SERS substrates in recent years. Some reports attempted a link between the particle morphology and SERS efficiency. A novel seeded growth approach was used to obtain surfactant free Au NSt [35]. The Au NSt were then coated with a silica layer. Selective etching of the silica layer gradually exposed the NPs spikes, and SERS was obtained for different effective silica coverages (Figure 1b). A strong correlation between the observed SERS intensity and heat losses calculated for the AuNSt structures was found.

1.2. Single-particle, dimers and controlled hotspots Single Au or Ag nanoshells on silica or polystyrene beads have attracted much attention in the SERS community. The composition and the thickness of the shell layer together with the material and size of the core offer several handles for geometrical tunability. These options offer a great potential for controlling the SERS enhancement efficiency as well as the optimal excitation wavelength. In addition, it also opens the opportunity for simultaneous sensing with multiple single NPs detection techniques, such as SERS and dark field imaging. It was reported that the variation in SERS intensities between single nanoshells also needed to be taken into account when exploring those particles as SERS substrates [36]. Ag nanoshells on polystyrene (PS) beads, coated with Nile blue A as Raman reporter, presented strong (single) particle-toparticle variations in SERS spectra. In fact, the long tail distribution of SERS intensities from single nanoshells resembled that of single-molecule SERS measurements, although the dye

12

concentration was far above the single molecule regime. It was stated that the long tail intensity distribution was caused by the random dispersal of Ag NPs aggregates on the shells. That report highlighted that particle-to-particle variation has to be taken into account for any future application of nanoshells in SERS (Figure 1c). There is another type of core-shell nanoparticles that has been enthusiastically explored in SERS community. They are dielectric shell isolated SERS active metal core NPs [46, 47], or shell-isolated nanoparticle-enhanced Raman spectroscopy (SHINERS) [45, 48]. In one recent attempt, Li et al. [47] reported the immobilization of catalytically active metal nanoparticles on silica shell isolated Au core, and its application for identifying the working mechanism for CO oxidation on PtFe and Pd nanocatalyst. The SHINERS approach have been intensely explored in several applications, and the interested readers are strongly encouraged to consult a recent comprehensive review on the subject in [46]. Generally speaking, self-assembled coinage metallic NPs onto certain type of solid support provide excellent SERS efficiency, reproducibility and stability. For example, Indrasekara et al. reported a self-assembled Au NSt onto a flat gold film with a Raman silent linker [49] that yielded good SERS sensitivity. A potential drawback of the self-assembly methods is that the hotspots are fixed due to the physical confinement of the NPs. It could be useful for some applications to have a collection of hotspots distributed on a surface that respond to external stimuli. This concept was achieved using poly(acrylamide-co-acrylic acid) hydrogel microspheres as the supporting scaffold for polyol-reduced Au NPs. The gap between the Au NPs, which modulates the hotspot strength, was adjusted by changing the pH [50]. NP-labels combined to two or more spectroscopic techniques (e.g. SERS and dark-field spectroscopy) may provide a wider set of information, improving the analytical performance of the method. For instance, dark-field imaging can quickly pinpoint deposited NPs on solid supports and then a SERS mapping can provide additional local chemical information. The connection between SERS enhancement, which is a near field effect, with dark-field light scattering, a far field effect, is still a topic of great interest in SERS. This relationship was recently explored using tailored hotspots prepared by direct deposition of Au NPs (of various sizes and shapes) on an Au mirror [51]. It was found that, despite the plasmonic coupling effect between Au NPs and the mirror surface, strong SERS signal was obtained from larger NPs. 13

Control over nanogaps in nanoparticles aggregates offers a distribution of hotspots in a 3D environment. This increases the chances of analyte molecules to find hotspots and generate a measurable SERS signal. Therefore, the precise control over NPs aggregation is one of the ultimate goals in SERS from colloidal suspensions. Several developments in this area were reported in the last few years. In all of them, a molecular linker, such as cucurbit[5]uril (CB[5]), for example, was used to modulate the inter-particle nanogap [37]. It was claimed that modification with CB[5] produces a repeatable, fixed, and rigid inter-particle distance of 0.9 nm, regardless of the concentration of CB[5] (Figure 1d). Time-resolved extinction spectra and SERS were used to monitor the controlled aggregation. Moreover, CB[5] served as both an internal calibration probe and a host for potential analytes. The specificity of CB[5] suggests that the proposed method could find broad applications in solution-phase SERS sensing. Similarly, cucurbit[6]uril (CB[6]) was also used as a linker for Au NP dimer formation in solution [52]. Au NPs were initially modified with cysteamine to impart positive charges to the surface. Then, the positively charged groups were linked to carbonyl groups at the portal of the CB[6] molecules. By carefully controlling the stoichiometric ratio between the cysteamine-modified Au NPs and CB[6], various degree of aggregation were implemented. Au NPs dimers with a 1.8 nm gap were found to establish the optimum condition for SERS measurements. CB[n] were also used for the control of the inter-particle gap in an evaluation of the contributions of the interactions between the NPs and the substrate surface [53, 54]. It was found that the inter-particle distance played bigger role in terms of affecting the SERS signal strength than the gap between the NPs and the substrate. The precise control over Au NPs aggregation achieved via special DNA structures, named ‘DNA origami’, was another considerable development in SERS from colloidal systems [55, 56]. This particular approach allowed single-molecule SERS detection [57]. Protruding strands from the DNA origami structure were used as arms for the assembly of DNA-coated Au NPs. Variable center-to-center particle distances, ranging from 10 to 40 nm, were realized. The largest relative enhancement in Raman intensity of 103 was found compared to single particles. Additional reports using DNA to control nanogaps for SERS explored systematic variations in particle sizes, shapes, compositions, as well as various inter-particle distances, and laser wavelengths [58, 59]. The best SERS performance was reported for Au-Ag core-shell nanodumbbell structures with inter-particle distances smaller than 1 nm. Meanwhile, for larger 14

particles, the SERS signal increased with the size of the Au core for a fixed inter-particle distance. DNA origami was applied for the controlled self-assembly of Au NPs with tunable gap distance upon optothermal agitation [60], during which two 40 nm Au NPs were connected by a 5 layers stack DNA origami. Probe molecules of interest were intercalated into the DNA layers. High laser power illumination was applied to trigger the DNA shrinking, followed by Raman measurement at lower laser powers. The gap distance was controlled in the range of 1-2 nm. The dependence of the SERS signal of the trapped probe molecule on the gap distance was found to be in good agreement with theoretical calculations. An universal DNA hybridization chain reaction (HCR) was implemented to induce Au NPs aggregation as a new approach for SERS detection of numerous analytes [61]. The recognition element, an aptamer, was employed to form a triple-helix structure with a single stranded DNA (called universal trigger, UT). The binding of analytes to the aptamer released the UT, which in turn acted as the bridge to trigger HCR of thiol terminated DNA hairpins adsorbed on silicon spheres dispersed in aqueous solutions. The HCR caused the formation of a DNA polymer, triggering the aggregation of Raman-labelled Au NPs, which led to strong SERS. This highly sensitive method is claimed to be universal, by considering that the protocol could be directly applied towards other analytes by careful selection of the aptamer. In addition, HCR amplified the SERS signal, driving the LoD down to single cell detection or pM levels. Controlled formation of end-to-end Au nanorods (Au NR) dimers for SERS applications have been accomplished using polymer coats [38, 62, 63]. Thiolated polystyrene polymers were used to modify the ends of the Au NR, and assembled end-to-end structures was achieved, triggered by a proper solvent (Figure 1e) [38, 63]. Phospholipids were later introduced into the solution to terminate the assembly, resulting in a large population of Au NR dimers [62]. Compared with Au NR monomers, the dimers showed a 50-fold SERS signal enhancement, and the stability of the colloidal system was excellent for more than 110 days.

1.3. Colloidal self-assembling and suspensions of large ordered structures Self-assembly of nanomaterials in large areas would be preferable for potential realistic applications of SERS. Large area offers a better probability to capture analytes from diluted 15

solutions and also provide a pathway for numerous measurements in different regions of the substrate for better quantification statistics. Self-assembly of centimeter scale Au NR patterns were achieved using a solvent evaporation (capillary flow) method [64]. Selective poly(ethylene glycol) (PEG) modification of either the transverse or longitudinal surfaces of Au NR led to highly ordered self-assembly. Either vertically or horizontally aligned Au NR structures were realized on PEGylated wafers from the solvent evaporation process. The assembled layers were tuned by changing of Au NR concentration. The resulting SERS substrates were highly reproducible, and the vertically aligned Au NR presented better performance (3-folds larger) when compared to the horizontally aligned ones. Although it is generally accepted that aggregated metallic NPs provides better SERS performance compared with monomers, it has been shown that this is not always the case [38, 63]. When the ends of Au NR were modified with thiol-terminated PS, through a ligand exchange process, either end-to-end (Figure 1e) or side-by-side aggregates, shown in Figure 2, of controlled lengths were achieved through exposure to different solvent compositions. A dimethylformamide (DMF)/water (20%) mixture led to end-to-end aggregates, shown by a characteristic red shift of the LSPR band. Although a non-linear behaviour was observed in SERS intensity versus the Au NR average aggregation number plot, strong correlation was found between the SERS intensity and the extinction product (product of the extinction at the laser excitation and at the Raman-shifted wavelength) (Figure 1e). The self-assembly of Au NR triggered by adding tetrahydrofuran (THF)/water mixture into the Au NR suspension in THF led to side-by-side aggregation, which was monitored by a consistent blue shift of the longitudinal LSPR peak. Surprisingly, the SERS intensity recorded during the controlled side-to-side aggregation showed continuous reduction, as shown in Figure 2. Finite-difference time domain (FDTD) calculations, performed to determine the electric field changes during the self-assembly, are also seen in Figure 2. It was found that the normalized sum of E-field actually decreased with increasing in side-by-side NR assemblies, due to cancellation of the radial component of SP modes. This study expanded the understanding of the interplay between geometry, assembly, and near-field optical properties of nanomaterials, which impacted, for example, in the use of SERS to probe near-field properties of plasmonic nanoparticles assemblies [65].

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Figure 2. Left, variation of normalized SERS intensity of assembled Au NR with time. Right, electric field intensity profiles produced via 3D-FDTD simulation for side-by-side assembled NR from Ref [63]. ©2012 American Chemical Society; used with permission.

1.4. Self-assembly of NPs on planar and other types of platforms SERS substrates fabricated by the deposition of metallic nanostructures on planar surfaces are alternatives for analytical implementations that do not involve measurement from colloidal suspensions. Planar SERS substrates are suitable for integration in miniaturized devices and microfluidics. Although direct metallic deposition or nanoparticle immobilization are the main approaches used in this area [66, 67], there has been important developments on both the deposition techniques and the nature of the planar surfaces. A novel protocol for generating SERS hotspots that uses NaCl as sacrificing layer was proposed [68]. A 400 nm NaCl sacrificing film was thermo-deposited onto a Si wafer. Then, in the same deposition chamber, 20-50 nm Ag films were added by electron beam evaporation. The Ag on NaCl film was then stored in ambient environment, leading to cracks promoted by interactions involving Ag, Cl-, and water (from the air). Finally, the cracked film was transferred onto a PDMS stamp for SERS applications. The thickness of the Ag film and the storage time were both keys for optimizing the SERS performance, since these two factors determined the gap-sizes between Ag particles. A relatively large area SERS substrate that is easily transferable onto appropriate planar surfaces was prepared using this method. Nanomaterials are known to self-assemble at liquid-liquid interfaces (LLI) and liquid-air interfaces (LAI), forming organized and, to some extent, controllable super-structures. LLI was used as a tool to explore optical properties, including SERS performance, of Au NPs and Au NR 17

in situ and in real-time [69]. The space between Au NPs was manipulated by removing the

citrate stabiliser in aqueous solution, and the SERS signal increased by ca. 20-fold as the average space among the NPs dropped from 30 nm to 1.9 nm. Similarly, the gaps between Au NR were decreased by adjusting the acidity of the medium. Red shifts of both transversal and longitudinal LSPR modes were observed as the gap decreased and a 40 times increase in the SERS performance was reported for the closed packed structure. Self-assembly of coinage metallic NPs at the water-organic solvent interface offers some advantages as substrate for SERS applications. For example, an assembled nanoparticle layer offered similar SERS performance compared to suspended aggregates, except with better stability [70]. In addition, the self-assembly process occurred in minutes, and no intricate procedure was required to transfer those layers to a solid support. However, a major issue accompanying self-assembled nanoparticles at the waterorganic solvent interface is the requirement of surface modification reagents, which can contribute to the Raman background and interfere with the SERS analysis. The use of tetrabutylammonium nitrate as promoter for citrate reduced Ag NPs assembly at a water/dichloromethane interface minimizes this spectral interference problem [70]. The SERS performance was shown to be stable for up to 20 h and, most importantly, the relative standard deviation of the SERS intensities, measured in a 1 mm2 area, was only 1.1%. Similar NPs assemblies at the LLI have been applied for the detection of various analytes, including heavy metals [71, 72], multianalytes [73] from different phases [74], and other chemicals [72, 75-80]. The NPs self-assembly at the LLI can be further optimized by either physical or chemical methods. Supramolecular polymers have been utilized as anisotropic templates for self-assembly of spherical Au NPs for SERS (Figure 1f) [39]. On the other hand, electrochemical potential has also been applied for the controlled adsorption of Ag NPs at the water/1,2-dichlorobenzene LLI, which improved the selectivity in SERS detection [81]. The self-assembly of Au NPs on a spherical oil-water interface has also been reported as an excellent SERS substrate [82]. The oil phase acted not only as the assembly platform, but also as the extracting agent for the analytes, as shown in Figure 3. In addition, it also produced narrow SERS fingerprint bands that were used as the internal calibration standard for quantitative SERS analysis, similar to their previous report on a cyclohexane/water LLI [77]. The authors claimed that sensitivity of the plasmonic metal liquid SERS platform could be optimized through proper control of the particle density. More importantly, the substrate was 18

quite variable, versatile, and self-healing in a way that a highly sensitive and reproducible SERS measurement could be accomplished in a cuvette. The proposed liquid-like SERS substrate can be used in combination with portable Raman spectrometer for field quantitative SERS applications [83].

Figure 3. A multiphase liquid-state SERS analyzer. Reversible O/W encasing for self-assembly of metal liquid-like GNR arrays is realized in a common cuvette[83]. © 2018 Nature; used with permission. Virus-like particle (VLP) was used as an unusual platform for the deposition of Au NPs. The VLP SERS substrate was implemented for bio-analysis of several species, including DNA fragments [84]. For instance, 78 nm VLP anchored a 3D self-assembled structure of Au NPs provided a 10-fold improvement in SERS from a capsid. Additionally, 0.25 ng µL-1 of a singlestranded DNA (ssDNA) fragment was detected in vitro using this substrate. Wrinkled thin films were introduced as an alternative method for the preparation of micro/nanosized hierarchical structures without the need for lithography. Shape memory polymers were the supporting substrate for the self-assembly of Au NPs [85]. SERS substrates were obtained by heating up the Au-modified polymer over the glass transition temperature, and applying either uniaxial or biaxial forces. Interestingly, the structural evolution of the wrinkled

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surface could be manipulated through the diameter, number of layers, and density of the Au NPs (“programmable wrinkled surface”). Gold nanoprisms (Au NPr) are known to have good SERS performance due to their sharp edges. However, the preparation of Au NPr usually needed hydrophilic protecting layers, such as Cetyltrimethylammonium bromide (CTAB), that difficulted their assembly onto large areas. A closed packed monolayer of Au NPr was obtained after a one-step modification of the NPs with Polyvinylpyrrolidone (PVP), followed by dispersion in ethanol-hexane mixture and transfer onto a water air interface [86]. The Au NPr film was then transferred onto a solid support by contact and used as SERS substrate. The exchange of the surface groups by PVP did not change the morphology of the Au NPr. Both the assembled pattern and the inter-particle distance were adjusted by simply changing the organic solvents and by the introduction of additives, such as dodecanethiol and oleylamine.

2. Fabricated structures on solid supports The fabrication of SERS substrates using top-down approaches to directly imprint nanostructures onto solid supports is another area of very active research in the field. In this section we will discuss some of these methods, including advances in approaches based on focused ion-beam (FIB) milling, electron-beam lithography (EBL), templating, and photolithography. The different methods have their unique characteristic in which can be beneficial for particular applications. This section will, for that reason, be divided into three subsections: advanced nanofabrication methods (FIB and EBL mostly); methods for the fabrication of large area substrates; and methods for the fabrication of low-cost substrates in a large area.

2.1. Advanced nanofabrication methods Advanced nanolithographic techniques, such as FIB and EBL, are top-down approaches for the fabrication of SERS substrates that enable precise patterning of thin metallic films. The use of these nanolithographic methods allows fine control over shape, size, as well as the relative distance and relative orientation of plasmonic nanostructures.

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Plasmonic anisotropic nanostructures constructed in regular arrangements present strong electric field polarization dependence. This property can be used to improve analytical selectivity, since polarization modulation can separate normal Raman of solution interferences from the surface interacting analyte [87]. In that sense, several regular arranges of anisotropic nanostructures have been pursued for SERS over the last years to explore the polarization properties of the substrates. Gold concentric ring arrays (Au CRA) were fabricated by FIB milling of a 50 nm-thick Au layer, and applied for the SERS studies of dyes [88], as illustrated in Figure 4. The curved nanostructures led to a “butterfly”-like polarization dependence of the SERS intensity, as it may be observed in the SERS mappings of the 594 cm-1 band of Nile blue for orthonormal polarizations presented in Figure 4C and D. This polarization effect was observed because the relative orientation of the concentric lines results in a slow spatial variation in the polarization dependence. The Au CRA substrate was shown to present, in concentrations as low as 200 nmol L-1 of Nile blue, the characteristic long tailed SERS intensity distribution observed in single-molecule SERS experiments.

Figure 4. (A) Optical microscopy of a concentric rings structure milled on a 300 nm thick gold film. The ring periodicity is 870 nm, and the strip width is 435 nm. The scale bar is 20 µm. (B) 21

Scanning electron microscopy image of the rings. The scale bar is 20 µm. 2D-mapping of the SERS intensity of the 594 cm-1 band for 1 µmol L-1 NBA on the Au concentric rings structure for the laser light polarized (C) in the y direction and (D) in the x direction, as indicated in panel A. The spectra were obtained at each 1 µm in both x and y directions. Reproduced from ref. [88] © 2012 American Chemical Society; used with permission. Plasmonic nanoantennas are commonly fabricated using FIB lithography [89]. For instance, successive layers of Au(100 nm), TiO2 (40 nm) and Au (50 nm) on glass were converted to nanoantennas by a mild power milling beam [90]. The resulting nanostructure consisted of two interacting 35 nm Au NPs with a 6 nm-gap, surrounded by a ring reflector on top of a TiO2 semiconductor layer. The bottom Au layer acted as a plane reflector preventing scattering losses from the substrate. Single-molecule SERS statistics using two analytes were recorded from this single hotspot for dye concentrations as low as 200 nmol L-1. Bragg rings were used to focus plasmonic waves onto central interacting stars in arrays of nanostructures fabricated by FIB [91]. The central nanostructures consisted of two nanostars forming a gap hotspot. Different materials (Ag, Ag/Au alloys or Au) were used in the fabrication and the best SERS performance was achieved by the Ag device excited at 830 nm. The influence of the geometric characteristics of the system on the SERS performance was probed by varying the nanostar sizes from 100 to 240 nm and the ring inner radius from 278 to 407 nm. The optimum SERS performance was also dependent on the excitation radiation. The smallest structure was optimal for SERS at 633 nm excitation, while other dimensions led to largest SERS efficiency at 830 nm. An inverted resist approach was introduced to prepare hollow Ag nanostructures by FIB [92, 93]. A resist layer was coated over silicon nitride and then exposed to an ion-beam to define the desirable pattern. The FIB fabrication allowed controllable height, inner and outer diameters of nanotubes that were subsequently covered by silver. A typical hollow Ag nanostructure, with periodicity of ca. 3 µm, produced strong SERS response for the detection of 1 µmol L-1 cresylviolet. However, no LoD or any other similar figure-of-merit was reported. A different approach to produce hollow gold nanostructures involved the use of commercial polycarbonate (PC) membranes with a range of pore diameter as template, followed by gold sputtering at room temperature [94]. The pore diameter determined the Au nanotubes diameter. The SERS 22

performance of the substrates was evaluated, and the largest nanotubes (400 nm) presented the best SERS performance. This was assigned to the large density of joined nanotubes, due to the coalescence of the pores of the template. The Au nanotubes were shown to detect, in specific high performance sites, rhodaming-6G (R6G) at 0.1 nmol L-1 level, a concentration that suggest SM-SERS detection. EBL fabrication allowed obtaining Au CRA with varying ring periods (from 500 to 830 nm) and inner central disk diameters (from 0.965 to 1.48 µm). Two configurations were fabricated: in the first one, top and bottom Au rings were grown on a Poly(methylmethacrylate) (PMMA) layer and the substrate presented coupled Au CRA; in the second one, the PMMA layer was etched away, so that the top Au layer was removed [95]. Changes in both period and inner disk diameters resulted in a remarkable effects on SERS intensities for the first configuration. The Au CRA with the smallest period and inner disk diameters presented the best SERS performance for benzenethiol detection when the laser was focused on the central ring. Additionally, the coupling between upper and lower rings revealed to have strong influence on the SERS performance, so that the coupled ring substrate presented an 8× increased SERS intensity in comparison to the etched Au CRA. The construction of plasmonic antennas has also been performed using a combination of regular lithography and EBL [96, 97]. The resulting SERS substrate was an array of Ag NPs dimers (gaps around 5 nm), surrounded by a silver nanoring. The hotspot between the two nanostructures presented polarization-dependent SERS performance. SM-SERS detection was reported for a large number of spots in the device. EBL-based nanoantennas were also proposed recently for the focusing of infrared radiation to enable high-performance surface-enhanced infrared absorption (SEIRA) [98]. These devices allowed an improvement of two-orders of magnitude for the SEIRA compared to previous reports. The production of 3D-plasmonic cavity platforms that act as SERS sensor was realized using a two-step EBL procedure. The EBL method was used to generate Au nanohole arrays (Au NHA) on a Ti-on-glass substrate modified with Au deposits. The final structure consisted of nanoholes on top of Au nanocones, separated by ca. 100 nm cavity [99]. The resulting 3D plasmonic cavity nanosensor arrays were shown to present a LoD for 4-nitrothiophenol below 100 amol L-1. 23

It is challenging to produce gaps smaller than 10 nm by EBL. The difficulty arises from the so-called ‘proximity effect’, which is related to back-scattered electrons that distorts the nanogaps. That effect has been circumvented to some extent by using numerical computation to plan the electron dose [100]. Another option to overcome this challenge involved the use of an electron transparent thin Si3N4 layer under an usual resist layer [101]. As a result, it was possible to create an array of interacting Au bowties (Au BT) structures that presented uniform interparticle distances variable between 6 and 24 nm. The maximum SERS intensity was obtained for the smallest period of repetition of the interacting bowties, 550 nm, for excitation with 785 nm. It was also demonstrated that the smallest homogeneous gap resulted in the largest SERS performance (for 550 nm period). The substrate with the best SERS performance was used to obtain good quality SERS spectra from a 10 nmol L-11,2-di(4-pyridyl)ethylene (BPE) solution. EBL was also used to create a pattern that was subsequently exposed to an ion-assisted aerosol lithography for the deposition of 3D Cu nanostructures [102]. The growth of the nanostructures occurred preferentially on Si exposed areas and upwards to overpass the resist layer height, resulting in anisotropic structures. The procedure was performed twice, leading to branched nanostructures containing several high-curvature regions that are suitable SERS hotspots. The effect of the upper structure on the SERS performance was evaluated. The intrinsic roughness can have significant effects on the SERS performance of deposited nanostructures fabricated by EBL molds and vapor deposition of metals. Sow et al. have shown that the SERS efficiency of ca. 100 nm wide gold stripes is strongly influenced by the intrinsic roughness of the metal deposited by vapor thermal deposition, to the point that the dependence on the E-field polarization direction (observed on post-annealed samples) was severely reduced [103]. The post-annealing of EBL nanostructures allowed the creation of Au nanocylinder arrays with controllable gap-distances, resulting in 5 % spot-to-spot variations in SERS intensity determined from a SERS mapping experiment [104]. The use of Ar+ sputtering of plasmonic materials has been explored for the fabrication of SERS substrates for at least two decades. The use of both regular and magnetron sputtering is based on the ionization of Ar gas, which removes material from a target composed of the desired material (the cathode) to be deposited [105]. In recent years, there has been a tendency of using sputtering procedures combined to other techniques, such as ion beam milling or templating, to 24

improve the control over shapes and sizes of the plasmonic nanostructures. Yang et al. applied the methodology using a thin layer of amorphous carbon deposited over a magnetron sputtered layer of silver on silicon or glass as morphology director [106]. The as-deposited carbon over Ag substrates were exposed to a tilted Ar+ ion beam for carbon striping, resulting in Ag nanoneedles with lengths in the 280-920 nm range. The nanoneedles were evaluated as SERS substrates using R6G as a molecular probe and applied to the detection of ketamine hydrochloride in concentrations down to 2.7 ppb in only 3 s. In a different approach, LnF3 nanoparticles (Ln = Nd, Sm, Eu, Tb) deposited on Si wafer were used to drive the morphology of magnetron sputtered Ag NPs, and the best SERS performance was observed for TbF3-AgNP [107]. The SERS efficiency was evaluated considering the LoD for three analytes, R6G, crystal violet (CV) and p-aminothiophenol (pATP), which were 10-14, 10-11 and 10-10 mol L-1, respectively. The progress described above clearly demonstrates the value of using advanced fabrication tools (FIB and EBL) to produce highly efficient SERS substrates. On the other hand, it is important to emphasize that those methods are time-consuming and expensive. Therefore, they are not appropriate for mass fabrication and routine analytical applications.

2.2. Methods for large area fabrication There is a trend towards the development of methods that result in deposition of plasmonic nanostructures over 1 cm2 or larger areas, with consistent shapes and sizes. The goal is to produce structures with reasonable (<20% RSD) spatial (over large areas) and sample-tosample variation in SERS intensities Those methods are mostly based on imprinting, selfassembly or soft-templating, so that many of them could be optimized for mass production of SERS substrates with a reasonable cost-benefit ratio. Nanosphere lithography, a technique that can easily produce 1.0×1.0 cm2 nanostructuring by drop casting layers of polystyrene beads as templates, is one of the most established procedures for large area patterning of SERS features. Although this method lead to relative large patches of organized structures, it is important to point out that grain boundary defects are also common. The long range order becomes more challenging as the size of the beads decreases. Modification to the classical nanospheres lithography procedure has been proposed in 25

recent years [108, 109]. The resulting polystyrene layer was subjected to reactive ion etching (RIE), so that the interstice between beads increased. Then, an Au layer was deposited on top of the polystyrene film and the beads were solubilized out of the substrate. The final result was a large area nanohole array (NHA) over an Au thin film. The SERS performance of the NHAs was tested at different incidence-angles of the excitation laser. The best SERS results were obtained for an Au NHA with 820 nm of periodicity and 500 nm diameter hole, excited at 785 nm at 10⁰ of incidence for 4-nitrobenzenothiol. The SERS efficiency at 10⁰ was 40% improved relative to the excitation at 0⁰. Another modification in the preparation of plasmonic materials over polystyrene microspheres involved the deposition of a silver overlayer, followed by atomic layer deposition (ALD) of an angstrom-scale thick Al2O3 layer, finishing with an additional Ag overlayer, leading to a stacked Ag/Al2O3/Ag layer covering a 2 cm wide circular glass area [110]. That material was subjected to ion etching, which partially removed the upper Ag layer, resulting in nanoring cavities and, after partially removing the Al2O3 layer, a thin air nanogap was left between the two Ag layers. The SERS performance of that substrate was evaluated using adenine as probe molecule, and a LoD of 76 nmol L-1 was verified. The reported LoD was one order of magnitude lower than for the well-known Ag film over nanospheres (Ag FON), which presented LoD of 524 nmol L-1, and improved over previous reported LoD for several Ag FON SERS substrates. Another example of recent Ag FON report indicated a relative standard deviation for SERS intensities over 10 samples of 9% [111]. Arrays of gold nanodimples (Au ND) were obtained by self-assembling of silica NPs dispersed in ethoxylated trimethylolpropane triacrylate (ETPTA) monomer spin-coated on 4 inches diameter silicon wafers [112]. After photopolymerization of ETPTA, the silica particles were exposed to RIE to decrease their radius followed by 5 nm Cr deposition for adhesion and additional sputtering of a 100 nm gold layer. The Au ND array covered the whole silicon wafer. The RIE exposure time was the optimization parameter for SERS performance. The SERS efficiency improved for longer RIE, mostly because the multilayer silica nanoparticles resulted in more complex motifs after longer RIE, leading to a larger number of high-performance hotspots. Soft lithography is another approach for the fabrication of large area SERS substrates. PDMS molds with arbitrary shapes in the micrometer scale have been used as containers for Au 26

NR [113]. The PDMS molds, patterned with micro-containers of several shapes and sizes, were applied on suspensions of surface-modified Au NR on different flat substrates (silicon wafers, glass slides or TEM grids). Au NR were subjected to capping ligand exchange from CTAB to (1mercaptoundec-11-yl)hexa(ethylene glycol) (MUDOL), a thiol bearing amphiphilic ligand that is known to induce smectic order during solvent evaporation. This new ligand led to improved order and packing of the Au NR deposited on the micro-containers. The procedure resulted in Au NR supercrystals, with controllable shapes and sizes in the micrometric scale. High performance and uniform SERS from the probe molecule CV, excited at 633 nm, was observed. Softlithography using (3-aminopropyl)triethoxysilane (APTES) modified PDMS molds was also employed for the generation of Au nanostructures using emeraldine-salt polyaniline (p-PANI) as the reducing agent in a wet chemical deposition approach [114]. The resultant flexible SERS substrates had their performance evaluated using the intrinsic SERS spectrum of p-PANI. Large area fabrication of interacting particles was also achieved by the use of a wrinkled PDMS layer that presented long (hundreds of micrometers) veins after they were treated by plasma oxidation. The wrinkled surface was then used for plasmonic nanoparticle assembly [115]. Au NPs were further modified by bovine serum albumin (BSA) and kept at a pH above the isoelectric point of the protein to ensure a negative charge for the nanoparticles. The BSA modification allowed highly packed Au NPs on the PDMS wrinkles, as well as the transfer of the nanoparticles from the polymeric template to quartz substrates as large as 1×1 cm2. The resulting plasmonic substrates were proposed as potential SERS substrates. Aligned block copolymers (ABCP) were shown to self-assemble in long nanowires using a solvent-assisted nano-transfer printing [116]. Those nanostructures were transferred to PMMA films; subsequently, Au or Ag films were deposited on the PMMA replica and then transferred onto a Si substrate, relying on the weakening of the interfacial interactions between the plasmonic material and the polymer replica by solvent vapors. Nanostructured surfaces as large as 2.0×1.5 cm2 were prepared and applied as SERS substrates. The deposition procedure allowed control over the formation of long nanowires or vertically stacked NR. To evaluate the SERS performance, detection of glucose in commercial contact lens was achieved at LoD of 100 µmol L-1.

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NR arrays of silver over zinc oxide were prepared using ZnO nanorods as templates, yielding SERS-active areas as large as 7.0×1.5 cm2 [117]. The ZnO nanorods were obtained by a wet method using ZnO seed deposited on a glass substrate, followed by hydrothermal reaction of zinc acetate in the presence of ammonium hydroxide. The resulting rods were covered by Ag using magnetron sputtering, and employed as SERS substrates that presented better performance than sponge-like Ag substrates. The use of anodized aluminum oxide (AAO) as a base for the electrodeposition of silver was modified by exposure of the Ag nanowires tips to an additional deposition of Au by magnetron sputtering [118]. The resulting substrate presented improved SERS performance compared to regular Ag nanowires fabricated using AAO. The batch-to-batch reproducibility over 20 samples was reported to be better than 4% relative standard deviation (RSD). Carbon nanotubes (NT) have been shown to be stackable in aligned, quasi-periodic superstructures, named super-aligned carbon nanotubes (SACNT) [119], which are claimed to be potentially mass produced. Al2O3-covered SACNT were transferred to 8 inches Si wafers and inductive coupled plasma (ICP) was used to transfer the template, using SF6 as the etchant gas and C4F8 as protector. After removing the SACNT layer, Au was deposited on the Si wafer by electron-beam, resulting in a quasi-periodic interconnected Au nanowires network over the wafer [120]. The fabricated substrate was shown to produce SERS spectra of R6G for concentrations down to 1×10-13 mol L-1, as well as good SERS response for the phosmet pesticide in concentrations as low as 1 nmol L-1, which is three orders of magnitude lower than the maximal residual contamination allowed for that pesticide. The reported SERS intensity variability over a 4 inches wafer was lower than 5%.

2.3. Low cost methods for plasmonic materials deposition over large area A challenge for the widespread use of SERS by non-specialists it the actual costs involved in the fabrication of a high-performance substrate. This limitation has encouraged several research groups to pursue low-cost fabrication alternatives for the deposition of large area substrates with outstanding SERS performance. Among the possible approaches, soft

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lithography and imprinting have particularly presented several developments recently in terms of both methodology and integration to microfluidics devices. Photolithography is a well-established method that can be implemented at low cost. 3DSERS substrates were obtained by the deposition of Ag on lithographically-modified substrates [121]. Patterned surfaces were exposed to 4-methylbenzenethiol (4MBT) to improve contact to Ag nanocubes (Ag NC) added using a Langmuir-Blodgett transfer method. The resulting SERS substrate was completely covered by Ag NC. SERS performance was evaluated using the Raman response of the 4MBT linker, and high homogeneity in SERS response over a large area was reported. The performance was dependent on the shape and size of the pyramidal microstructures defined by photolithography, as well as their inclination relative to the substrate. The last variable proved to be the most relevant for the optimization of the SERS performance. Deep-UV lithography has been proposed to prepare 300 mm Si wafer for Au deposition [122]. The sensitization of the photoresist was performed twice using a 90o rotation, so that 90 nm dots (nanopillars) were constructed. Au was sputtered on the nanopillar array, and the thickness of the Au film strongly influenced the resulting gaps between the coated posts, modulating the SERS performance. The device presented improved reproducibility and performance over the commercial KlariteTM SERS substrate (Notice that the Klarite was one of the first SERS substrates ever commercialized. However, as several other commercial SERS substrates followed suit, the Klarite product line was discontinued in 2017). However, a quite relevant claim is that the scalability of the proposed methodology is the most striking achievement, as the batch-to-batch repeatability over three runs presented very consistent SERS intensities.

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Figure 5. (a) SEM image of the large area nanograting substrate. (b) White light diffraction picture of the gold-coated µ-arrays. The inset is the SEM image of one of the µ-arrays. The blue scale bar is 1 mm. (c) The average SERS spectra of 4-mercaptopyridine (adsorbed from 10 mM solution) (red) TM/perpendicular polarization and (blue) TE/parallel polarization against the nanogratings main directions. Polarization directions are illustrated in the figure. Excitation: 633 nm. Adapted from ref. [123] © 2017 Elsevier B.V. Used with permission. Laser interference lithography has also been used to fabricate nano-grating that presented strong polarization dependence [123, 124]. It has been demonstrated the possibility of producing both large areas, up to 1 inch2, and µ-arrays of nano-gratings with variations in geometrical parameters of less than 5%. Typical gratings are presented in Figure 5(a), and a µ-array is presented in Figure 5(b). The polarization dependence of the substrate was demonstrated by both LSPR measurements as well by exciting the SERS spectrum of 4-mercaptopyridine at two perpendicular polarization directions relative to the grooves of the gratings (Figure 5(c)). The µarrays were utilized as SERS sensor integrated into a microfluidic device for the quantification of 8-quinolinol, an environmental pollutant. The polarization dependence allowed the separation of contributions to the SERS spectra of species adsorbed on the plasmonic substrate from the Raman signal of molecules that are not adsorbed and, thus, are a non-polarization dependent background. 30

The use of nanoimprint lithography (NIL) is a good alternative for the fabrication of large area SERS substrates [125]. The methodology involved gold-deposited nanodisks over a Si wafer. The Au nanostructures were further grown, aiming at controlling their interstitial nanogap. The resulting nanostructures were shown to present average gaps as small as 5 nm. The small gap led to considerable improvement in SERS performance and homogeneous spatial distribution of intensities in 16×16 µm2 SERS mappings. The “pen-on-paper” principle is an approach to construct SERS substrates that is becoming more common recently. The concept is based on the use of an ink made of plasmonic materials that may be transferred directly onto paper, leading to a flexible and high-performance SERS substrates with arbitrary trace geometry [126]. The approach is versatile, as different shapes, sizes and composition of plasmonic nanoparticles may be employed, as long as the suspension is stable at reasonably high concentrations to function as an ink. Some of the plasmonic nanoparticles used in this approach included spherical Ag NPs and Au NPs, as well as Au NR. Papers loaded with any of those NPs types have shown high SERS performance and stability. The substrates were shown to be able to detect malachite green, R6G and the pesticide thiabendazole in concentrations down to 1×10-10 mol L-1, 1×10-12 mol L-1 and 0.02 ppm, respectively, with a 10-20% spatial variability in SERS intensity. A variation of the pen-on-paper method is the ink-on-paper, which involves the use of commercially available inkjet printers as the deliverer of the plasmonic material onto paper [127]. Plasmonic ink transferred onto chromatographic paper was used as substrates and good quality SERS spectra were recorded using a portable Raman spectrometer. The examples of applications included the use in lateral flow experiments, dipsticks sensors and swabs. Using regular optics for the excitation/collection (instead of a Raman microscope) allowed for probing a larger area and averaging over several spots which reduced the SERS variability. The swab approach was shown to allow the detection of the thiram fungicide by SERS in quantities as small as 10 ng spread over a small glass area. The impregnation of Zari fabric by Ag NPs is a rare example of large area fabric for SERS applications [128]. The combination of the metal coat characteristic of Zari fabrics with Ag NPs, which was incorporated in three diverse ways, resulted in SERS substrates that were evaluated using 4,4’-bipyridine as the probe molecule. The substrates presented high SERS 31

intensity for the probe-molecule, and it was used to obtain the SERS of adenine in concentrations as low as 0.01 mmol L-1. Rose petals have also been reported as supporting platform for SERS. The hydrophobic surface of the petals enabled the concentration of plasmonic NPs into a small area after solvent evaporation [129]. The microstructures of the rose petals served to concentrate both the Ag NPs and the analyte, as well as to increase inter-particle coupling that improved the SERS performance. The SERS LoD for R6G from a Ag NPs on rose petal substrate was as low as 1×10-15 mol L-1. Real time fabrication of SERS substrate on copper, using an electrochemical deposition was also reported[130]. The use of pure water as the electrolyte and copper (or brass) as working electrodes led to the formation of copper oxide nanoparticles on the electrode surface within minutes The copper oxide-coated surfaces were shown to perform well as SERS substrates. Surprisingly, Ag did not show the same effect.

3. Advances in SERS quantification Reliable quantification of the SERS results is essential for the application of the technique in analytical chemistry. Methods for the generation of consistent calibration curves are constantly being reported and there are several interesting examples of reliable quantification procedures using SERS. The introduction of chemometric methods [131-134] into the SERS data evaluation has intensified in the last years and the application of strong statistical analysis is a step forward towards realistic implementations of the technique as a routine method in analytical laboratories. Quantitative SERS is now routinely achieved in immunoassays [21, 135-138]. Although SERS has many advantages, such as high sensitivity and the capability of analyzing multiple analytes simultaneously, one major issue for its application in immunoassays is still the variability in SERS signal. Several techniques for fine control of the geometry of metallic NPs have been reported [139, 140]. Therefore, SERS variability due to size and shape have been tamed to a certain degree. Another source of variability is NP-NP interactions. A statistical approach, which enables the sampling of thousands of NPs aggregates, was established to 32

quantify this type of variability. SERS probes consisting of PEG-coated NPs, coated with Nile blue, and modified with streptavidin as the recognition element were used. The SERS probes were used to map a biotin modified gold slide. The statistics based on the correlation between the distributions of SERS intensities under each laser illuminated spot and the distributions of SERS probe cluster sizes in the same area (probed by SEM) was evaluated. It was found that there was a simple polynomial relationship between the SERS probe cluster size and the resultant SERS intensity. Thus, by simply controlling the probe incubation time, and limiting the distribution of cluster sizes, quantification of targeted sites could be performed. Suspensions of plasmonic NPs in liquid phase are widely used for various SERS applications, yet they perform poorly in terms of signal reproducibility and quantification. The poor reproducibility is attributable to the reduced stability of the NPs in suspension and their dynamical optical properties. Internally etched Ag@Au@SiO2 NPs [141, 142] were developed by first synthesizing plasmonic NPs and coating them with a silica layer. Then, the silica layer was carefully etched with concentrated NH4OH solution, leading to a membrane that covered the Ag@Au NP core. Aromatic thiols were then able to diffuse into the membrane and adsorb onto the metal core. The advantages of the Ag@Au@SiO2 as a SERS substrate included good electromagnetic stability of the NPs (due to the spacer effect of the membrane) and limited SERS active volume [142]. The SERS signal variation with concentration correlated well with an adsorption isotherm. It is possible that such kind of SERS substrates could find wide application in SERS quantification directly from NPs suspensions. Although most of the efforts reported in literature to improve the reproducibility of SERS measurements focussed on controlling the quality of the SERS substrate, it was recognized that any SERS quantitative analysis might suffer from under-sampling [143]. The role of the sampling size (i.e., the size of focused laser spot relative to the number of NPs being probed in a typical assay) on the spatial distribution of SERS intensities was evaluated in an immunoassay and correlated to computer simulations. It was found that the sampling bias can be minimized by increasing the laser spot to sample a larger area. In this sense, the best configuration for SERS immuno-assays involves a lower magnification lens in combination with higher power laser to decrease the sampling problem.

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Most literature reports in SERS intend to improve quantification by promoting increase in the SERS signal. However, the SERS background is also an important issue and need attention for proper quantification. Recently, a “snow jet” technique (snow jet is the mechanochemical process used in microelectronics to remove dirt and organic contaminants from surfaces) was used to investigate a commercial KlariteTM SERS substrate modified with benzenethiol. The goal was to explore the effect of the snow jet on both the SERS signal and background [144]. The mechanochemical perturbation led to an average SERS signal increase of ~500%, while the background decreased following the snow jet cleaning. This suggests that SERS signal and background are from different origins, the latter being more dependent on the morphological changes of the substrate. That report indicates that different optimization parameters might be required to improve (decrease) SERS background.

3.1. Quantification through large area mapping Two problems that limit reliable quantification are generally identified in SERS applications: the inhomogeneous nature of the substrate; and the random distribution of the analyte throughout the substrate surface. A quantification strategy based on large area SERS mapping have been introduced to counter those effects [145]. Mapping the total area of the analyte spread at the surface provides better relative standard deviation than a simple average of a few randomly chosen points. This strategy over-performed the conventional point-averaging protocol particularly well at lower concentrations. An obvious drawback for this large area method is the significant increase in the analysis time. This problem has been tackled in modern Raman microscope systems that use a combination of line focussing and rapid detection systems to decrease the mapping time. The uniformity of the Raman enhancement efficiency has been probed for Ag deposited on Si and PDMS [146]. The Si scaffold was fabricated through chemical etching, while the PDMS was prepared by a top-down nanofabrication method. Considerably large variances in sizes and shapes of the nanostructures for the Si-based substrate were found. Although the Sibased platform showed larger SERS signal, the homogeneity of signal was not great, as expected for a substrate that presents large sizes and shape variations. On the other hand, the PDMS-based substrate performed better in terms of repeatability, although it presented smaller Raman enhancement efficiency. 34

3.2. Quantitative SERS near the single molecule regime The SERS response at very low surface concentrations shows strong intensity fluctuations [147]. This behaviour has been attributed to the dynamics of a small number of molecules visiting a SERS-active hotspot. At very low surface concentrations, the chance of having many analyte molecules within the laser illuminated area is small. Taking the scarcity of the highly enhancing SERS hotspots into account, the chance of having the analyte molecules on the hotspots is even smaller. In extreme cases, the observed SERS signal from the illuminated area is believed to be originated from a single molecule adsorbed at one hotspot. Since the properties of the hotspots varies widely (spatially and within a population), the SERS signal presents strong fluctuations (SERS intensity fluctuations – SIF). The SIF lead to large error bars in calibration curves determined by direct measurements of the absolute value of the (average) SERS intensities. The quantification in this case might not be straightforward, since the strong fluctuations could affect the reliability of a calibration curve. Therefore, in the conditions of strong fluctuations, more advanced techniques for data analysis must be implemented for quantification. An effective protocol for quantitative SERS analysis in the strong fluctuation regime based on statistical and Fourier analysis has been reported [148]. Au NPs were selfassembled onto a microfluidic channel and the Raman scattered photons were recorded with an avalanche photodiode at certain frequencies. A comparison of the Raman signal of the probed molecules to the background photoluminescence of the Au NPs was used to construct a Freundlich isotherm model, leading to quantification down to the single molecule detection regime. Alternatively, Fourier analysis based on the temporal fluctuations led to a more accurate analysis, since the analysis of the fluctuations in the frequency domain appear to be independent of the variability of the hotspot strength.

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Figure 6. (a) Plot of the average values of the non-negative matrix factorization with alternating least-squares algorithm (NMF-ALS) at 95% confidence level versus surface density of the analyte (enrofloxacin); (b) Calibration curve build with digitalized SERS mapping for enrofloxacin at ultralow concentration. [149] ©2018 American Chemical Society, used with permission. Another approach for quantification of analytes at ultralow concentrations (lower than 1 nmol L-1) was recently demonstrated [149]. In Figure 6a one may notice that the average values of “SERS intensities” (in fact, output from chemometric methods, specifically non-negative matrix factorization with alternating least-squares algorithm - NMF-ALS, were used to obtain coefficients that are equivalent to “SERS intensities”) present low sensitivity towards the surface density of the analyte. This illustrates the issue of a small number of hotspots with widely distributed SERS performance in the presence of the highly diluted enrofloxacin analyte. It is clear from Figure 6a that at such low concentrations the calibration curve cannot be used directly for quantification. However, since any SERS signal intensity originates from a single molecule, the absolute value of the SERS intensity is not important. Each SIF event represents a single molecule being detected. Hence a simple digitization procedure can be implemented and a calibration curve can be generated by just counting the number of single molecule events detected in a Raman mapping. Figure 6b shows a revised calibration curve obtained by plotting the number of single molecule events against the number of molecules per unit surface area. The analytical curve presented in Figure 6b for extreme low concentrations present a good degree of linearity. This digital SERS method allowed the quantification at ultralow concentrations of

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antibiotic contaminants in water. It should also be noted that this method requires SERS substrate with reasonable SERS intensity spatial variations (<20% RSD).

3.3. Other examples of quantitative SERS Another exciting area of development may be found in the integration of SERS detection with analytical separation methods. A sensitive quantitative high-performance liquid chromatography (HPLC) with SERS analysis technology was reported. The method is based on sheath-flow hydrodynamic focusing and internal calibrations [150]. Several model analytes, including thiamine, folic acid, and riboflavin, were introduced into the HPLC system and detected by UV-Vis and SERS detectors in tandem. The sheath flow configuration greatly enhanced the SERS sensitivity, while using the solvent (acetonitrile) as an internal standard improved the repeatability of the analysis. A high level of reproducibility was observed even for data obtained several days apart. Classical quantification methods, such as internal standard, have also been used in SERS. Tan et al. [151] reported the decoration of multilayered graphitic magnetic nanocapsules (AGNs) with Au NPs and its application as a reproducible SERS substrate. It was found that the graphitic nanomaterials present a unique Raman band that located in the Raman silent region of the biomolecules. Thus, the Raman band of the graphitic nanomaterials at 2655 cm-1 could serve as an internal standard for quantitative SERS measurements. The authors found that, with the internal standard, the RSD% could be as low as 6.8%. The same group also reported GrapheneIsolated-Au-Nanocrystal (GIAN) [152] for accurate and rapid SERS analysis. GIAN is chemically inert, does not produce background fluorescence, and also allow graphene bands to be used as internal standards, which significantly improved the calibration curves. The authors applied the substrate for the quantitative analysis of crystal violet in fish muscle and scale. In another report, Rhodamine B (RhB) in chili oil has been quantitatively determined by thin layer chromatography (TLC)-SERS [153] using melamine as an internal standard. Extracts from chili oil containing RhB and RhB standard were both spotted onto a TLC plate. After development, the silica gels containing the sample and the standard were removed and dissolved in acetic acid solution. Regular citrate-reduced Ag NPs were mixed with the analyte and dried on aluminum for SERS measurements. The standard RhB spot served as both a position mark as

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well as a reference for the amendment of the calibration curve. RhB in chili oil was determined using both a pre-built calibration curve and single point calibration.

4. Advances in SERS substrates integrated to devices The integration of SERS substrates into several devices have been attracting attention due to the increasing need for on-site and/or remote sensing based on spectroscopic techniques. The initial development of such integrated devices was slow for a long time due to sizing and sensitivity limitations, but in the last decade, fast-paced innovations has been encouraging for both microfluidics and optical fibers-based devices. This section will review some of the most relevant recent development on approaches for device integration of SERS for analytical applications.

4.1. Integration of SERS substrates on microfluidics devices The integration of SERS substrates to microfluidics devices is an exciting approach, due to the multiplexing possibilities that this integration enables. SERS detection is ideal for microfluidics architectures due to the miniaturization and flexibility of the devices, as well as the requirements for minimal sample amount and multiplexing possibilities [154-158]. A direct approach for the integration of SERS substrates in microfluidics involved the mixing of Ag NPs to the analyte in the inlet of the microfluidic device followed, after the mixing step, by concentration of the Ag NPs/analyte in a sensing region, from which the SERS spectra were obtained using a portable Raman spectrometer (Figure 7a) [155]. The reported LoD for the food contaminant melamine was 63 ppb and for the fungicide Thiram was 50 ppt. In a different approach, electron-beam lithography was used to produce assemblies of Au nanotriangles integrated in microfluidic devices [159]. The system was then used for the label-free SERS detection of ochratoxin-A. A similar procedure was used for the detection of levofloxacin with a LoD of 0.8 µmol L-1 in Ag NPs loaded microfluidic device [160]. The detection of multiple analytes was proposed using a plus sign-shaped device that allowed introducing samples from different sources into the flow along with Ag NPs [161]. This procedure resulted in multiplexed detection of several dyes in concentrations down to the nmol L-1 range.

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Microfluidic plasmonic devices were used for SERS detection of methamphetamine in saliva. The application of principal component analysis allowed an automated classification of the SERS spectra [162]. Additionally, the detection of unlabelled DNA sequences was achieved using a competitive displacement procedure in a microfluidic device. The displaced probe was a strong SERS active molecule, which was put in contact with Ag NPs and detected by SERS [163]. The procedure was also extended for multiplexed detection of DNA.

Figure 7. a) Optofluidic SERS microsystem with packed microspheres for passive concentration, an integrated micromixer to promote adsorption of the target analyte, and integrated fiber optic cables for optical excitation and collection[155]. ©2017 American Chemical Society, used with permission.; b) Photographs of fabricated centrifugal microfluidic SERS platform, adapted from ref. [166]. ©2017 American Chemical Society, used with permission.; c) Cutaway illustration of material flows in the free-surface microfluidic channel. The aqueous microfluidic phase flows from left to right (blue arrows). The gas phase flows from back to front (green arrows). Analyte molecules (red spheres) diffuse from the gas phase into the liquid phase (red arrows). Nanoparticles (white spheres) suspended in the aqueous phase adsorb to suspended analyte 39

molecules before interrogation by 658 nm laser light (red vertical beam) for detection by SERS[167]. ©2014 SPIE, used with permission; d) Centrifugal microfluidic disc with open Raman spectroscopy measurement chambers [170]. ©2017 Royal Society of Chemistry, used with permission.. The technique of mixing SERS-active nanoparticles suspended in microfluidic flows was expanded for the detection and quantification of cancer cells at low concentrations [131, 164]. This procedure, supported by chemometrics, led to discrimination between SERS-labelled normal and cancerous cells. Cells populations were discriminated as well as quantified. The possibility of constructing SERS-immunosensing devices based on the immobilization of surface-modified Au NSt on microfluidic channels was also explored [165]. The modified Au NSt held the SERS-tag for the experiment. The modification involved the bonding of specific antibodies to the Au NSt, which allowed multiplexed SERS sensing of several breast cancer biomarkers in patient-mimicked human serum by conjugation of a specific antibody in each microchannel. Si nanopilars obtained by RIE covered by Au layers deposited by electron beam evaporation were utilized in the fabrication of microfluidic devices on centrifugal microfluidic discs (Figure 7b) [166]. The SERS active Au nanopilars obtained by that methodology were applied in the detection of melamine spiked on milk samples. The detection was based on the wicking effect filtration, which resulted in regions that concentrated unwanted macromolecules and regions were the analyte could access that the nanostructures and were used in SERS detection. The estimated SERS LoD for melamine was 10 ppm. It has also been proposed the preparation of Au nanostructures immobilized on microfluidic devices by means of etching of an Au/Ag alloys. The process was modulated by PS beads, followed by removal of the bead and dealloying. The procedure resulted in disks of porous Au nanostructures [167]. The integrated device (Au nanoporous arrays immobilized in a PDMS microfluidic channel (Figure 7c)) was tested for the SERS detection of R6G solution, with a LoD of 5.16 nmol L-1, as well as for monitoring the SERS intensity time evolution. The LoD for SERS detection for dopamine was 32.4 nmol L-1 and for urea 0.67 mmol L-1, which are both within physiological relevant ranges.

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A free-surface microfluidic device was applied for the detection of airborne organic substances using the SERS technique [168, 169]. The device was built so that part of the microfluidic path was exposed to the environment. Free flowing Ag NPs were the SERS-active materials, ensuring continuous refreshing of the active substrate. Initially, the analyte vapor, 1 ppb 2,4-dinitrotoluene, was directed to the headspace above the open surface of the device (kept at dew temperature), followed by SERS spectral acquisition. The SERS signature of the analyte started being observed after 2 min of exposure, and reached maximum intensity after 6.5 min [168]. The possibility of using a microfabrication-free microfluidic device was also explored. In this case, an Ag NPs flow was directed into a gas-phase analyte flow. The resulting segmented two phase flow travelled a determined distance before reaching the Raman detection point. The SERS detection of 750 ppb 4-aminobenzenethiol was reported using this method [169]. Centrifugal microfluidics devices allow reliable analyte manipulation and placement in the channels. The presence of free-surfaces in these microfluidic devices eliminated spectral contamination by the polymeric components (PMMA) in SERS experiments (Figure 7d) [170]. The preparation of open surfaces for Raman was optimized by considering the forces acting during rotation, and resulted in a highly reliable procedure as long as the ideal rotational conditions are met. The device was used to study contaminations in milk by melamine. The reports were based on doping melamine on full cream milk, and resulted in LoD of 203 ppm for the contaminant, without any interference from PMMA. Large-area SERS substrates were fabricated on glass slides using dielectrophoresis to form a chain of Au NPs interconnecting gold strip electrodes that were 1 µm apart [171]. The chains of Au NPs were formed by drop-casting a suspension of Au NPs in the presence of the analytes, followed by the electric field induction of Au NPs chain formation. The SERS spectra of benzenethiol and adenine were obtained for concentrations as low as 100 fmol L-1. Figure 8(a) to (c) shows an approach for the application of SERS-active optical fiber tips as the sensing elements for in-line analysis in microfluidic devices. The optical fibers were positioned after the mixing region in the microfluidic device, so that no plasmonic material was introduced in the flow path (Figure 8a) [154]. The SERS optrodes were inserted in the microchannel through small hole punched mechanically through the PDMS. Figure 8(d) shows a 41

sequence of 5 different dyes introduced in the device and measured in sequence after a simple cleaning procedure. The proposed devices also allowed the multiplexed detection of mixtures in different concentrations (Figure 8(e) and (f)), as well as the real-time monitoring of time dependent processes.

Figure 8. (a) Schematic representation of the microfluidic gradient chip. Two different analyte solutions at the same concentration were simultaneously pumped into the microfluidic chip through inlets 1 and 2. At the straight portion of each channel (shown by arrows), small holes were punched with a syringe needle and the SERS optrodes were introduced. The other ends of the optrodes were linked to a Raman microscope; (b) labeled picture of the experimental setup 42

for on-chip multiplex detection; and (c) schematic of the fiber optrode tip sensor with nanoparticles and linker. The core and clad size of the fiber used in this report are 62.5 mm and 125 mm respectively. (d) In-line monitoring of the flow of analytes: (i) R6G (10 µM), (ii) CG (5 µM), (iii) oxa (5 µM), (iv) NBA (5 µM), and (v) CB (10 µM). All spectra are background

corrected and offset for clarity. The numbers on the spectra indicate the main vibrational features (in cm-1) from the respective dye. The x-coordinate (Raman shift) for the spectra i, ii, and v is at the bottom of the graph; and the x-coordinate (Raman shift) for the spectra iii and iv is at the top of the graph. The solutions were introduced into the channel sequentially from i to v (from bottom to top, as shown by the arrow). Note that R6G and CG are not in resonance with the laser and their spectra are due to SERS, but the other three dyes are also in resonance (SERRS effect). (e) Average spectra from each channel of the dilution chip; inset shows the enlarged portion of the spectral region where bands from each dye in the mixture can be uniquely identified. At 682 cm-1 (inset), top to bottom, spectra are from outlet channel 1 to channel 5, respectively. (f) SERRS fractions of both dyes plotted against their respective concentrations in each channel. The error bars show the variation over three measurements in the same channel. Adapted from [154]. © 2012, The Royal Society of Chemistry. Used with permission.

4.2. Integration to optical fibers and microcapillaries based devices Integrating plasmonic nanostructures on optical fiber tips produces the so-called SERS optrodes (Figure 8c), which are very promising devices for miniaturization, integration and field applications [172, 173]. Several advances in this area were reported in the time span covered by the present review. The most relevant goal of plasmonic nanostructured optical fiber tips is usually to allow remote sensing using SERS. It has been proposed the modification of optical fiber tips by Au NPs clusters selfassembled by deposition from reverse micelles of poly(styrene-block-2-vinylpyridine) [174]. The SERS performance was evaluated both in remote configuration, in which the excitation of SERS occurs in the fiber tip opposite to the nanoparticle modified region, as well as in a ‘direct’ configuration, where the SERS spectra is acquired directly on the nanoparticle modified region. The remote configuration resulted in SERS intensities 36% larger than those obtained for 43

isolated Au NPs modified optical fibers. The nanospheres lithography technique has also been applied to modify optical fiber tips for SERS sensors [175]. That report also indicated the possibility of immobilizing various periodic gold micro-features, which resulted in four structural motifs. The SERS sensitivity measured from those tips were similar to previously reported optical fiber configurations. The immobilization of Ag NC on the tips of optical fibers was achieved by immersing a freshly cleaved multi-mode optical fiber to a suspension containing Ag NC and an analyte of interest, so that a meniscus was formed and induced the self-assembly by laser irradiation through the fiber [176]. The laser induced self-assembly was driven by fast evaporation of the solvent in the meniscus under irradiation. The presence of the analyte was used to monitor the deposition kinetics by SERS, so that a saturation time was determined. The reported SERS LoD for pATP was 0.1 nmol L-1. The SERS detection of gases using microcapillaries fabricated by a capillary drawing system was achieved by modifying the capillaries surface by the polycation poly(allylamine hydrochloride) (PAH), which adhered electrostatically to citrate-stabilized Au NPs [177]. The lateral walls of the Au NPs-modified microcapillaries exposed to organic gases (pyridine or 4nitrophenol) were directly excited in a Raman microscope. The kinetic dependence of the SERS intensity was investigated, indicating that pyridine reached adsorption equilibrium 5 s after exposure, but 4-nitrophenol needed as long as 600 s to reach equilibrium. The large difference was attributed to differences in intermolecular interactions. A modified procedure to fabricate ultramicroelectrodes for SERS involved attaching Au NPs to the microtips of glass nanopipettes, resulting in both high conductivity and SERS activity [178]. The SERS spectra were acquired by external excitation from the positioned microcapillary under the laser light. It was demonstrated that it was possible to obtain SERS spectra of 10 mmol L-1 4-mercaptobenzonitrile. It was also demonstrated the detection of intracellular glutathione, by inserting the capillary tip into single HeLa cells using micropositioning setups.

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5. Advances in analytical applications Efforts to improve the analytical performance for dilute samples and complex mixtures have been among the hottest topics in SERS research recently. The recent development of (super)hydrophobic substrates and direct sampling capabilities, among others, are driving those advances toward new limits for the SERS applicability. In this section, we will review some of the applications recently reported in the literature on super/ominihydrophobic substrates and approaches on direct sampling.

5.1. Concentrating solution ominiphobic substrates

phase

samples

with

(super)hydrophobic

and

Local concentration of analytes by (super)hydrophobic SERS structures to improve sensitivity and LoD have been reported by several groups [179-186]. The proposed analyte concentration mechanism is straightforward: on a (super)hydrophobic SERS surface, the contact line of the liquid sample droplet cannot be pinned during solvent evaporation. As a result, solutes in the droplet will be carried over by the capillary flow towards the interior of the droplet and eventually accumulate on a very small area. This means that the local concentration of analytes could be greatly increased using this approach. For instance, a concentration factor of 104 was reported for a nanofabricated superhydrophobic SERS substrate [179]. SERS detection of rhodamine dyes even at attomol per liter concentrations was achieved. Hakonen et al. [187] reported a new and very interesting type of superhydrophobic SERS substrate. They used mask-less lithography combined with reactive ion etching for the preparation of Si/Au nanopillars. Water droplets on the substrate initially demonstrated rolling, as usually seen in regular superhydrophobic surfaces. However, shortly after that observation, the pillars in the substrate clustered together due to the penetration of water and showed strong adhesion to the droplet. Thus, the substrate not only demonstrated the concentrating effect as seen in regular superhydrophobic SERS substrate, but also showed greatly improved SERS efficiency due to the clustering of the nanopillar. Femtomoles of nerve gas were successfully detected by SERS using this substrate in combination of a handhold spectrometer. A drawback of (super)hydrophobic SERS substrates is that they only work for aqueous solutions, since the substrates repels only water. For samples containing organic solvents, the 45

analyte concentration effect is not observed. In order to solve this issue, an universal platform that works for both aqueous and non-aqueous solutions, called slippery liquid infused porous surface-enhanced Raman scattering (SLIPSERS), was reported [182]. In that approach, the platform was named omniphobic, i.e., it repelled both aqueous and non-aqueous liquids. This was achieved by infiltrating a nano-textured Teflon® membrane (pore size of 200 nm) with a perfluorinated liquid. Then, liquid samples were placed on the surface, along with SERS-active NPs. The platform allowed the detection of several analytes, including environmental pollutants and biological species in different solvents. One practical issue related to the concentration approach based on solvent repulsion is that the final dried spot is generally very small (for example, ~ 30 µm diameter [179]). Therefore, it can be difficult to find the dried spot, even with the help of an optical microscope. This inconvenience was overcome by using a directed concentration concept [188]. The setup included a superhydrophobic substrate, which served as the water repellent platform, and a SERS active fiber optrode as a fishing rod. Initially, the SERS optrode was inserted into the spherical aqueous droplet of the analyte deposited on the superhydrophobic surface. The sample droplet shrunk during the evaporation of the solvent, until it was eventually caught by the SERS-active hydrophilic optrode, carrying the concentrated analyte. The SERS measurements were accomplished at the distal end of the fiber, making it suitable for fast measurement and remote sensing. The benefit of the superhydrophobic concentration step was roughly 3 orders of magnitude in analyte accumulation.

5.2. SERS substrate with direct sampling capability For surface contaminants, such as pesticides on agricultural products as well as explosives and drugs on luggage and clothing, the conventional sample preparation for SERS would involve collecting samples, preparing solutions, and applying them to a SERS substrate for analysis. This approach is not time efficient and greatly hinders the application of SERS in field analysis. The SERS community has recently been developing substrates that could directly collect a sample onto a Raman enhancing media. Liz-Marzan et al. summarized the recent development of flexible SERS substrates that can be used in this sort of applications [189]. Flexible SERS substrates may be highly sensitive, reproducible and relatively low-cost, making them ideal for direct sampling in field applications. 46

200 nm-thick silver-coated sandpaper have been used for direct sampling [190]. Commercial 3000 mesh sandpaper provided the optimum roughness for the best SERS performance. Figure 9 summarizes how this substrate was used to swab the pesticide triazophos from pears, tree leaves, and glasses. A SEM imaging of this type of SERS substrates is shown in Figure 9a. Even at 53.3 pmol cm-2 triazophos on pear, the SERS signature of the contaminant was still clearly visible. The advantages of the sandpaper-based SERS substrate included very low cost (less than US$0.10 for a pad of 5×5 cm2) and the ease of mass production fabrication.

Figure 9. (A) Swabbing test on a whole pear. The inset shows SEM of the SERS swab; (B) SERS spectra of triazophos obtained by swabbing different surfaces. (a) Pear (~0.3cm2, 53.3 pmol cm-2); (b) tree leaf (~0.6cm2, 266 pmol cm-2); (c) plastic (~3cm2, 10.5 pmol cm-2); (d) glass (~4cm2, 4.2 pmol cm-2); (e) 5uL 1ppm triazophos. The arrows show the most dominant bands of triazophos. © 2014 Elsevier, used with permission.

Filter paper-based SERS swab has also gained attention from the community [191-193]. The SERS substrates developed using such approach have been applied, for example, in the swab detection of R6G and malathion [191] on glass surface. A SERS substrate prepared by stamping was used for the swab detection of benzocaine, a pain reliever, on glass surfaces [194], although no “real-world” samples were tested. A simple method for the fabrication of flexible SERS substrate based on filter paper [195, 196] involved the direct immersion of a piece of filter paper into an Au NR suspension for 2 days, followed by drying with a nitrogen flow. The authors 47

demonstrated the detection of less than 140 pg of 1,4-benzenedithiol (1,4-BDT) residue spread over 4 cm2 by swabbing the glass surface. Au NPs were deposited on PMMA templates by self-assembly from toluene/water interfaces [197]. The PMMA was first dissolved in toluene and then added to water. Since the toluene phase was less dense, it remained on the top of the water layer. The evaporation of the toluene solvent led to a thin PMMA/Au NPs residue on top of the water surface. Finally, a Polyethylene plastic film was used to lift the PMMA/Au NPs composite. The resulting transparent flexible SERS substrate was used to detect malachite green on fish skin. Another example of flexible SERS supported on polymer consisted of an assembly of Au@Ag NC at water/air interface that was transferred onto PDMS [198]. The resulting flexible substrate was used to stamp and examine illicit drugs on banknotes without any requirements for additional sample pre-treatment. The efficiency of the direct sample collection approach is dependent on how well the target analytes transfers to the collection surface [199]. Thus, three-dimensional substrates, such as Q-tip/swab, may have advantages, since they may support a larger load of analyte relative to a (2D) sensing surface. In fact, swab sampling is one of the most established methods for surface contamination analyses due to its convenience and efficiency. The swabbing procedure has been used for detection of bacteria on mechanical ventilators [200] and textiles [201], as well as explosives [202]. It has been proposed a method for the fabrication of a SERS swab/Q-tip, based on self-assembly of Ag NPs on cotton swab followed by in situ enlarging [203]. Firstly, the cotton was modified with (3-aminopropyl) trimethoxysilane (APTMS), cured in nitrogen at 120 ⁰C, and immersed in Ag NPs suspension for 30 min under sonication. Later, the Ag NPsmodified cotton swabs were soaked in silver nitrate and then in NaBH4 solutions under sonication to promote the in situ growing. Finally, the substrate was subjected to curing in nitrogen at 120 ⁰C. It was found that the SERS Q-tip was capable to detect femtograms of the Raman probe NBA, and 5 ng of the explosive DNT from glass surfaces through direct swabbing. This substrate may have broad applications in food safety screening as well. SERS substrates constructed in hydrogels are another type of platform for direct sampling. For instance, a commercial hydrogel used in soft contact lenses was mixed with 2,3dihydroxypropyl methacrylate to form a copolymer hydrogel [204]. Later, the copolymer 48

hydrogel was soaked in a mixture of DMF and water containing EDTA. Then, the gel was cut into pieces and put on places of interests, sitting for various times from 30s to 4 h, depending on the targets. Finally, concentrated Ag NPs were applied on the gel to enable SERS analysis. Although this procedure yielded good results, the methodology is cumbersome, since it requires Ag NPs to be added later in the process. An alternative procedure involved the direct mixing of Ag NPs suspensions with agarose [205-207]. The agarose hydrogel SERS substrate was tested for the detection of dyes from various materials, including wool, silk, and cotton. Ag NPs dispersions mixed with agar-agar were also tested [206]. Both detection and extraction of dyes were performed by cutting the Ag NPs-agar gel matrix into small cubes, and placing one face of the cube in contact with the textile surface. The final extraction step was to wet the system with a drop of ethanol. After 30 min of extraction, the gel was left to dry and SERS analysis was performed. It should be noticed that in most reports the hydrogel served either as supporting media to help improve the sensitivity of SERS analysis [208-210] or as simple extraction media [204]. Furthermore, these substrates are difficult to apply for fast screening on-site, because of the time needed for the analysis, which ranges from 30 min [207, 210] to 2 h [208]. A new substrate was introduced to address these drawbacks. Inspired by the properties of polyvinyl alcohol (PVA) hydrogel, commonly known as “toy slime”, a novel shape adaptable SERS PVA hydrogel was developed [211]. The schematics for the use of that substrate is shown in Figure 10. Firstly, an Ag NPs suspension was prepared using sodium citrate as reducing reagent. The Ag NPs suspension was then used to dissolve PVA powder at 90 °C in a vigorously stirred water bath for 2 h. Later, the mixture was concentrated by centrifugation, re-dispersed, and mixed with appropriate amount of sodium tetraborate to yield the final “slime” SERS substrate (Figure 10). The as prepared substrate is a non-Newtonian fluid that slowly flows with its own gravity. It was also found that the final concentration of Ag NPs in the “slime” played a very important role in the SERS performance. Interestingly, the Ag NPs concentration in the “slime” was found to be much higher than the one obtained by direct synthesis. It was shown that, at optimum conditions, the Raman probe NBA at 0.79 pg/~3 cm2 could be successfully detected through the stick and peel process described in Figure 10. Additionally, the “slime” SERS substrate was used to detect Raman probes at ng levels on diverse surfaces, from 400 mesh sandpaper surface, to cotton

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fabric, and plastic films. The “slime” SERS substrate was also explored for the detection of illicit dyes and pesticides at ng level.

Figure 10. The preparation of the slime SERS substrate and SERS of the surface contaminant with the shape adaptable substrate,(a) AgNP–PVA mixture; (b) sodium borate; (c) SERS slime; (d) wood stick; (e) SERS slime on a wood stick; (f) contaminated surface; (g), SERS of the surface contaminant [211]. © 2014 Royal Society of Chemistry, used with permission.

The application of foam-like 3-D SERS substrates has been further expanded by using chitosan [212] and PDMS as a supporting scaffold [213]. A PDMS sponge was easily obtained by simply using sugar as a pore forming reagent. The PDMS sponge was used to adsorb Ag NPs. It was found that this PDMS-based SERS substrate was not only able to extract minute amount of pesticides, such as triazophos and methyl parathion on fruit peels at ng level, but also to detect those pesticides in juices with minimum requirements for sample pre-treatment. In addition, the analyses were realized in just a few minutes (ranging from 3 min for surface analysis to ~30 min for solution samples). This kind of substrate could find wide application for on-site food safety screening.

6. Advances in bioanalytical applications Biomedical and bioanalytical applications continue to be a main trust of the SERS research. A significant amount of progress was reported in the last few years on the development of new materials and devices that enables SERS from biologically relevant systems. Those developments were mostly in the SERS bioanalysis directly within cells and bioassays in body 50

fluids, which will have dedicated subsections here, but several other aspects of the bioanalyses have also been touched by the SERS research, which will be discussed in the 6.3 subsection.

6.1. SERS bioanalysis within cells New approaches for the utilization of SERS-based analysis directly on cells have been recently proposed. It would probably be fair to establish that the main challenge in this area is the compatibility and stability of the plasmonic material exposed to the biological medium. Bioconjugation is an essential step, as well as surface modification to impart site selectivity for SERS diagnostics and imaging. The use of bioconjugated plasmonic vesicles from amphiphilic polymer brush coated with Au NPs for SERS-based cancer targeting and drug delivery was one of the recent advances [214]. The reported plasmonic vesicles could play several roles, including as SERS-active plasmonic contrast imaging. SERS imaging with plasmonic vesicles was achieved by specifically labelling them to target cancer cells while monitoring intracellular drug delivery. SERS imaging of cancerous cells was also performed using a three steps Au@Au NPs that presented a ca. 1.1 nm inner nanogap. The gap was created from modification of the initial Au NPs with thiol-modified poly-adenine and a SERS reporter (either 4,4′-dipyridyl or 5,5′dithiobis(2-nitrobenzoic acid)) and additional Au deposition [215]. The resulting Au@Au NPs presented a narrow size-distribution around 76 nm. The detection of living cancer cells was performed after modification by hyaluronic acid, which chemically recognized the CD44 receptor, a tumor surface biomarker. PEG-coated Au NPs have been used to SERS-label three distinct cell surface markers on LY10 lymphoma cell line [216]. The PEG modified Au NPs were functionalized by monoclonal antibodies. The specificity of the functionalized Au NPs were demonstrated by performing SERS experiments either directly on colloidal suspensions or using confocal SERS mapping. The approach was finally expanded to allow the collection of SERS directly from labelled cells in a commercially-available flow cytometer. The SERS spectra of either Staphylococcus aureus or Escherichia coli were reported to present a marked decay in their characteristics vibrational bands when in the presence of antibiotics [217]. The SERS spectra were obtained from biofilms of the bacteria over Ag NPs 51

modified mesoporous Al substrates. The strong dependence to the presence of antibiotics was used to rapidly perform minimum inhibitory concentration (MIC) and antibiotic susceptibility tests (AST) of several samples, including clinical isolates. The SERS results presented good agreement with more traditional pharmacological methods, such as the agar dilution method. Lectin-modified Ag NPs were designed and used to detect the expression of carbohydrate species on cell surfaces [218]. Carbohydrate-lectin interactions were demonstrated for three different lectin species and three different cell types. Because glycans are overexpressed in cancerous cells, the approach was expanded to discriminate cancerous from noncancerous prostate cells using confocal SERS mapping of sialic acid, a key glycan in cancerous cells. The SERS mappings allowed discrimination between cancerous and noncancerous cells more clearly than fluorescence microscopy. It has been shown that the excitation wavelength impacts strongly on the information provided by SERS experiments in red blood cells. This is due to changes in interaction of the plasmonic NPs with hemoglobin [219]. It was claimed that the molecular information from the SERS spectra is highly dependent on the material and size of nanoparticles in contact with red blood cells, which directly impacts on the possibility of comparing results for different types of NPs.

6.2. Bioassays for DNA and antigen/antibody recognition in body fluids An important approach for early diagnosis of several diseases, and particularly cancer, is based in the analysis of biomarkers in body fluids, such as saliva, urine, and blood. Some exciting developments of SERS-based methods for early diagnostics based in analysis of body fluids have been reported within the time lapse covered by this review. As a matter of fact, Au NPs specially labelled by 5,5’-dithiobis(succinimidyl-2-nitrobenzoate) (DSNB) in pH=8.5, followed by antibody immobilization and surface blocking by BSA, were developed for the SERS detection of two pancreatic adenocarcinoma biomarkers in sandwich bioassays [138]. The sandwich bioassay consisted of constructing capturing lines of antibodies in delimited Au layers over glass. The gold pattern formed small wells and were modified by dithiobis(succinimidyl propionate) (DSP) followed by the chemical binding of the antibodies anti-MMP-7 in the first capturing line (row) and anti-CA 19-9 in the second row. Anti-MMP-7 and anti-CA 19-9 are 52

both monoclonal antibodies specific for pancreatic adenocarcinoma. These two rows were used for calibration and an additional ‘patient’s sample’ row was also present. The patient’s sample row had part of the well modified by anti-MMP-7 and other part by anti-CA 19-9. The surface was blocked from non-specific binding by a nonfat milk/Tween 20 blocking buffer in PBS. The proof-of-concept sandwich SERS-immunoassay consisted of exposing the two calibration row of wells to the specific antigen (at several concentration levels) in diluted human serum, and the patient’s row received diluted de-identified patient serum samples from healthy males, which resulted in capturing the antigens onto the substrate. The captured antigens were then exposed to the Au NPs modified by antibodies, and the SERS responses were recorded. The SERS LoD for MMP-7 was 2.28 pg mL-1 and for CA 19-9 was 34.5 pg mL-1 in the calibration rows, which was claimed to be one order of magnitude lower than for ELISA. The use of silica shell-isolated Au nanoparticles (Au SHIN) was proposed for the development of immunoassays employing both surface-enhanced fluorescence (SEF) and SERS [220, 221]. The proposed approach is summarized in Figure 11. As seen in Figure 11 the Au SHIN (100 nm Au core + 4 nm silica shell thickness) were covered by the SERS reporter NBA, followed by an additional coating of a new ultrathin layer of silica up to a total shell overlayer thickness of 10 nm. The silica surface of Au SHIN was functionalized by Zika monoclonal antiNS1 antibodies (Zika-mAb), resulting in SERS nanoprobes. The immunoassays (represented in Figure 11) involve the modification of glass cover slides through EDC/NHS chemistry to immobilize Zika-mAb. The modified cover slides were exposed to different clinically relevant concentrations of Zika NS1 antigen and exposed to the SERS nanoprobes. The exposed surfaces were SERS mapped over a 40×40 µm2 area, and the resulting chemical maps were analysed and correlated to the concentration of Zika NS1 antigen. The proposed method presented a LoD of 12.5 ng mL-1 for the Zika NS1 antigen, which compares favorably to previous reports at 75 ng mL-1.

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Figure 11. Schematic illustration of (a) Zika-mAb-SERS nanoprobe assembly: Au-SHIN (∼100 nm Au core + 4 nm silica shell thickness); Au-SHIN + NBARaman reporter layer; Au-SHIN + NB Raman reporter layer + final ∼10 nm silica shell (SERS nanoprobe); conjugation onto Zika NS1 monoclonal antibodies (Zika-mAb). (b) SERS immunoassay platform for detecting different concentrations of Zika NS1. The platform is irradiated with 632.8 nm laser line and the SERRS signal from NBA molecules, located in a close distance of gold nanoparticles (∼4 nm), are recorded by area mappings. Brighter spots indicate higher intensity of the NB band at 593 cm−1. Adapted from ref. [221].© 2018 American Chemical Society; used with permission.

The modification of lysine residues by NHS in antibodies was used as a strategy to improve the binding to citrate-capped Au NPs, forming stable conjugates in the 6.0 to 8.5 pH range [222]. The modification decreased the positive charges in the protein, preventing aggregation of the Au NPs. The efficiency of the modification was dependent on the lysine content of the antibodies. Immunoassays performed with NHS-modified Au NPs indicated that

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the antigen binding functionality was maintained, and improved SERS performance to detect and identify the H3 avian influenza virus was reported. A SERS-based immunoassay that used antigen-mediated aggregation of Au NPs was developed [223]. The resulting Au NPs aggregates were concentrated on a nanoporous membrane, and then taken for SERS analysis. The detection limit of the proposed assay, 1.9 ng mL-1, is one order of magnitude lower than the one obtained using ELISA (35 ng mL-1), and the assays could be implemented in serum. Effective detection of tuberculosis (TB) chemical markers is essential to improve the effectiveness of the diagnostics. A SERS-based immunoassay to detect TB was developed as a sandwich assay. The SERS label were 60 nm Au NPs modified with DSNB, followed by adsorption of mannose-capped lipoarabinomannan (ManLAM) monoclonal antibodies (mAb) and the capture platform was obtained by adsorption of DSP followed by ManLAM mAb on glass-supported 200 nm thick gold layers [136, 224]. Although ManLAM is found in TB patients, there is an important drawback to its use as a diagnostic marker: ManLAM form complexes with other proteins in the serum which significantly decrease the sensitivity of an assay. The proposed SERS approach used protein denaturation by acidification with HClO4 to disrupt the ManLAM-protein complexes. The acidification procedure significantly improved the LoD down to 2 ng mL-1 for the ManLAM, compared to a 500 ng mL-1 LoD for the untreated samples. The proposed bioassay was extended to human serum specimens of 24 TB-positive and 10 healthy patients, resulting in reliable response in 22 of 24 TB-positive and in all 10 healthy patients. The ability to increase the density of efficient SERS hot-spots is fundamental for the development of highly sensitive immunoassays. To increase hotspots density and efficiency it was proposed adding a SERS tag during the reduction process in the synthesis of the NPs [225]. An aniline derivative (N-(3-amidino)-aniline) (NAAN) further coated the Au surface. The oxidation of NAAN led to poly(N-(3-amidino)-aniline) (PNAAN) that acted as a self-assembly directing agent for the formation of Au superparticles (Au SP). The proposed approach allowed labelling Au SPs with up to four SERS reporters. The SERS reporters featured spectral fingerprints that were different enough to allow identifying each one uniquely in a mixture of the

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Au SPs. An immunoassay for the detection of the staphylococcal enterotoxins B using Au SP was reported and the achieved LoD was 0.08 pg mL-1. Ag NPs conjugated to maghemite (γ-Fe2O3) magnetic NPs were modified with SERS tags and specific antibodies for each of three bacterial strains [226]. The resulting SERS nanotags were concentrated by magnetically removing bacteria from suspension, and specific strains were identified based on the unique fingerprint of each SERS tag. The approach allowed detection of cell concentrations as low as 10 colony forming units per mL (CFU mL-1) of E. coli, S. typh and MRSA. Each strain was detected simultaneously and uniquely discriminated based on PCA analysis of the SERS results. SERS was also coupled with rapid vertical flow (RFV) immunoassay technology for fast diagnostics suitable for point-of-care [227]. RFV devices were prepared using 13 nm Au NPs modified with protein A, followed by pATP and blocked by BSA. The modified Au NPs were deposited on nitrocellulose test strips, which sustain a high load of the NPs due to the vertical flow. The enhanced NPs loading resulted in low spot-to-spot variations in the SERS intensity of pATP. The detection of a mAb for Hepatitis C biomarkers was performed and the minimum concentration that resulted in measurable SERS intensity was 53.1 µg mL-1.

6.3. Analysis of nucleic acids by SERS DNA and RNA fragments are among the most important analytes targeted for SERS detection. The analysis of DNA from real samples by SERS normally includes denaturation, fragmentation, separation, amplification and sensing [228]. Although SERS could produce both quantitative and structural information from minute amount analytes, its application to direct DNA analysis can be challenging. For instance, it is difficult to obtain SERS from double stranded DNA because the phosphate moieties on the DNA prevent close adsorption onto a negatively charged coinage metal NP surface [228]. It was proposed that the modification of Ag NPs with spermine, a positively charged polyamine, would promote NP aggregation, producing a strong SERS signal. As a proof of concept for this approach, the sensing of a hybridization event, as well as examples of SERS recognition of single base mismatches and base methylations (5methylated cytosine and N6-methylated adenine) in duplexes were reported [228]. 56

Quantitative label-free SERS detection of DNA, both single and double stranded, [229] was achieved by adding KI and Mg2+ into citrate reduced Ag NPs. The modification of the Ag NPs surface by iodide ion not only “cleaned” impurities from the metal surface, but also prevented the direct interaction of DNA with the surface. The latter effect was important to improve the reproducibility of the method. In addition, Mg2+ helped with the aggregation of Ag NPs, thus enhanced the SERS signal. Additionally, vibrations from the phosphate backbone of the DNA polymer were also detected and used as internal reference for the quantification of the bases. SERS of DNA has been utilized for the quantitative and simultaneous detection of three bacterial meningitis pathogens [230]. The analytical concept involved a sandwich hybridization process, where the DNA was labelled with both a SERS active dye and biotin. The latter was used as a handle for the separation of target DNA through magnetic beads. Then, λ-exonuclease, was used to break the separated DNA and release the SERS dye. Quantification of multiple bacteria was accomplished through the quantification of the SERS dyes response. A methodology to use SERS for the detection and quantification of transgenicity from genetically modified plants was proposed [231], based on the conjugation of ssDNA to SERS tags modified Au NPs. The recognition and quantification of transgenicity of DNA involved simple hybridization to target complementary DNA fragments. The quantification of target copy was achieved by generating an analytical curve using thiolated ssDNA for each target. The SERS sandwich assay allowed a LoD of 0.1 pg of the transgenic DNA.

6.4. Other bioanalytical applications A method to determine the localization of Au NPs by quenching the SERS of dyeconjugated nanoparticles that have not been interiorized by cells has been presented [232]. The quenching was caused by tris(2-carboxyethyl)phosphine, a well-known fluorescence quencher for cyanine type dyes, that is not able to penetrate cell membranes. The possibility of monitoring living cells secretion using SERS was explored using a glass capillary (500 nm at tip) modified with Au NPs. The modified tip was placed in contact to the extracellular environment, positioned at ca. 30 µm from the cell walls [233, 234]. The SERS sensor was shown to probe cellular secretions with single-molecule sensitivity. Selective detection of pyruvate, lactate, adenosine triphosphate and urea was demonstrated, and a distance 57

dependent gradient of extracellular species was correlated to the changes in SERS intensities at different positions. Specifically for neurons, it was also demonstrated the possibility of monitoring dopamine, glutamate, γ-aminobutyric acid and acetylcholine, including the response of the living cells to external chemical stimuli [234]. Ag NPs modified by lipid bilayers were employed to mimic cell walls, allowing the study of the secondary structure of Amyloid-β40 (Aβ40) oligomers attached to the lipid bilayers by SERS [235]. Information on Aβ40 oligomers structure may be essential to the understanding of Alzheimer’s disease. The SERS results, backed-up by solid NMR results to ascertain the presence of β-sheets, indicated that the Aβ40 was in a near-orthogonal orientation of the H-bonds in the backbone, quite different from less toxic mature Aβ fibrils. That information could provide structural basis for the mechanism of Aβ40 toxicity. Another example of lipid bilayers modified Au NPs was reported for the detection of the cholera toxin (CT) [236]. The approach was based on the selective coupling of plasmonic materials in the presence of CT. To achieve selectivity to CT, monosialoganglioside (GM1) was embedded to a lipid bilayer, along with a SERS-reporter dye. In the presence of CT, the interaction with GM1 resulted in aggregation of the Au NPs, and the SERS of the reporter was, consequently, the analytical signal used to characterize the presence of the toxin. SERS was used to study the interaction between the soluble apoprotein LasRLDB and modulators of P. aeruginosa quorum sensing (QS) [237]. To understand the interaction, it was necessary to orient the binding of the LasRLDB protein to Au NPs immobilized layer-by-layer SERS substrate. Specifically, it was necessary to perform genetic modification to the C-terminus of the polypeptide by introducing a cysteine to the structure, which resulted in a strong Au-S interaction and led to controlled conformation on the Au surface. SERS experiments probed specific conformational changes of LasRLDB upon interaction with modulators of P. aeruginosa QS. It has also been shown that hybrid materials containing plasmonic components may be used to detect signals from QS in biofilms of P. aeruginosa using pyocyanin as a signaling molecule for QS [238]. The approach consisted of employing cell-compatible hybrid materials. Specifically, macroporous poly-N-isopropylacrylamide (PNIPAM) hydrogels loaded with Au nanorods (Au NR@PNIPAM) were used for SERS detection in colonized and non-colonized regions of the substrate. Mesoporous TiO2 thin film over a submonolayer of Au nanospheres, Au@TiO2, was used to a specific detection of pyocyanin in biofilm-colonized surfaces by SERS 58

mapping the QS

vibrational signature. Moreover, mesoporous silica-coated micropatterned

supercrystal arrays of Au nanorods (Au NR@SiO2) enabled plasmonic detection of pyocyanin at early stages of biofilm formation and imaging of the phenazine produced by small clusters of bacteria colonizing micrometre-sized plasmonic features. 3-mercaptophenylboronic acid modified Au NPs were used as SERS probe for hydrogen peroxide secreted by living cells [239]. The molecular probe was oxidized to 3-hydroxyphenol in the presence of H2O2, and the SERS spectra presented characteristic changes proportional to the peroxide concentration. The ability to detect H2O2 was also used to probe glucose in artificial urine and normal human serum by coupling to the action of glucose-oxidase, an enzyme that catalysis the conversion of glucose to gluconic acid and H2O2. Another method based on the chemical modification of the SERS probe was developed for the detection of gaseous aldehydes in the breath that are considered markers of lung cancer [240]. Dendritic Ag NPs were modified by pATP and taken into contact with gaseous aldehydes. The interaction with aldehydes resulted in the formation of an imine on the Ag surface; a chemical change that was monitored by SERS. The spectral changes related to the formation of the C=N bond correlated to the concentration of the aldehydes and they were dependent on the aldehyde structure. The reported LoD for this method was 2 ppb, and presence of water vapor did not produced any marked influence in the results. GIANs has also been applied for the label free detection of bilirubin in blood serum of newborns [152]. When the liver function of a newborn becomes impaired, the bilirubin concentration in blood rises. Thus, it is important to both detect bilirubin and quantify its level. GIANs has shown strong affinity to bilirubin owing to the hydrophobic graphitic shell. When the GIANs are combined with cellulose paper strip, the bilirubin in blood serum could be separated and detected. Moreover, since the graphitic shell can act as internal standard, quantification of bilirubin was naturally realized. Spatially-offset SERS (SESORS) was implemented to tissue analogs in back-scattering geometry [241]. Commercially available NPs were spin-coated onto glass slides. Pork belly tissue analogs of various thicknesses, ranging from 1.35 to 8.10 mm, were used to cover the NPs films. The tissue analogs were placed over the NPs film, and by spatially offsetting the second probe relative to the first led to improvements over the change in axial focus in a single optical

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probe experiment, a result that could be used in the further development of SESORS as a noninvasive method to analyse tumours in vivo.

7. Other applications Although biomedical applications constitute one of the main focuses for the SERS research community, several other areas of applied analytical chemistry, including food, environmental and forensic sciences have experienced significant developments. The SERS technique is constantly being tested as a replacement for several traditional analytical approaches. For example, Lopez-Lopez et al. reported the analysis of gunshot residues (GSR) by SERS [242]. Usually, GSR analysis is performed by mass spectrometry or ion mobility spectrometry. Au NPs were used as SERS substrates for the analysis of smokeless gun powder as well as GSR particles. SERS has also been utilized for the analysis of inorganic ions using different strategies. It is well known that metal ion chelating reagents, such as the chromophores used in spectrophotometry, could be used in SERS analysis of metal ions. 4-(4-phenylmethanethiol)2,2’: 6’,2’’-terpyridine (PMTTP) was synthesized as SERS probe, where the terpyridine moiety worked as the chelating reagent, to demonstrate the concept of ion selective optrode [243]. Metal ion binding to the PMTTP-modified optrode led to changes in the SERS spectrum of the ligands. Those changes were reversed when an optrode tip was exposed to EDTA, which removed the bound metal ions. The SERS spectra of PMTTP in the presence of cadmium ions at different concentrations were recorded and a calibration curve was constructed. Cadmium in water was semi-quantitatively determined at a LoD of 100 nmol L-1. The use of salen ligand for the SERS detection of Ni(II), Cu(II), Co(II) and Mn(II) was also reported [244]. The lowest LoD was found to be 1 µg L-1 for Ni(II). In another strategy, DNAzyme has been used for the detection of heavy metal ions [245]. The double stranded structure of the specially designed Pb2+-specific DNAzyme was disturbed due to interaction with Pb2+ ions. Thus, when the distal end of the DNAzyme was labelled with a Raman probe, the initial minimum signal (due to distance from the surface) increased remarkably upon the presence of Pb2+. An extremely high sensitivity of this approach (LoD = 8.9×10−12 mol L-1) on a silicon wafer-supported Ag-core-Au-satellite SERS substrate was reported. SERS with 60

functionalized Au NSt was applied in the determination of uranyl [246]. The Au NSt were modified with carboxylic acid terminated alkanethiols to facilitate the adsorption of uranyl. A LoD of 120 nmol L-1 was achieved. SERS has also been utilized for in situ monitoring of catalytic reactions. One of the most exciting reports in this area was the use of SERS for monitoring metal catalyzed chemical reactions at the single molecule level [247]. The approach involved immobilization of 4nitrobenzenethiol (4-NBT) between a thin layer Au film and Ag NPs. Laser illumination promoted photochemical reactions that were probed by changes in the SERS spectra. The yield, dynamics, and magnitude of the photochemical processes of single molecules were explored. Au NPs immobilized in filter papers were also used as catalyst as well as an in situ, real time, reaction monitoring platform based on SERS [248]. This system was used for the monitoring of the conversion of 4-NBT to 4-aminophenol. SERS active filter papers were fabricated using simple methods and they were used for the determination of several analytes [184, 249-252]. For example, in the case of thiram and ferbam, the filter paper was made hydrophobic by an alkyl ketene dimer treatment [249]. Then, an Ag NPs suspension was dropped onto the filter paper, forming SERS-active hotspots. It was shown that the hydrophobic modification of the filter paper improved both the sensitivity and the repeatability of the analysis. A semi-quantitative determination of the antibiotic sulfamethoxazole, with LoD of 2.2 nmol L-1, for contaminated tap water was achieved in a microfluidic system integrated on an EBL-fabricated SERS substrate [253]. A combination of SERS and multivariate curve resolution was used for the detection of malathion on fruit peel [134]. The simultaneous detection of various aminoglycoside antibiotics was successfully achieved using a combination of LSPR and SERS [254]. The proposed dual detection method is suitable for the analysis of clinical human serum samples. An interesting new effect, called photo-induced enhanced Raman spectroscopy (PIERS) was introduced. PIERS is an additional improvement over the now traditional SERS driven by a photo-initiated electron transfer. The technique seems useful for the detection of various analytes, including explosives and biomolecules [255, 256]. The PIERS substrate consisted of citrate reduced Au or Ag NPs deposited onto a TiO2 rutile surface formed by chemical vapour 61

deposition. The analyte of interest was deposited on the metal nanostructure and the whole system was subjected to UV illumination to promote electron transfer from the TiO2 to the metal and the SERS was simultaneously recorded using a visible laser. The PIERS effect proved an additional one order of magnitude enhancement compared to regular SERS. Advances in the combination of SERS and electrochemistry (electrochemical SERS - ECSERS) for analytical applications have also been reported in the last few years. For instance, ECSERS was used for the detection of 6-thiouric acid in synthetic urine samples [257]. The SERSactive electrode was fabricated by depositing citrate reduced Ag NPs concentrate on commercial screen-printed carbon films. A LoD of 1 µmol L-1 was achieved by using a bench-top Raman spectrometer and a USB potentiostat. Simultaneous Raman and electrochemical measurements of neurotransmitters in a microfluidic chip demonstrated that molecules with the highest surface affinity showed highest SERS signal [258]. Among the strategies to improve the SERS detection sensitivity, there were several interesting reports on advances in the chemical modification of the substrate surface [259, 260] and/or magnetic-induced concentration of analytes onto the SERS substrate [261-264]. Hydrodynamic focusing approach has also been suggested to direct the analyte to the SERSactive surface [150, 265-267]. The detection of the 20 proteinogenic L-aminoacids [266], and eight biologically-active peptides [267] adsorbed on vacuum deposited Ag on anodized aluminum filter was achieved using the later approach. The simultaneous detection, identification, and localization of proteinaceous binding media found in artworks with Au NPs dimer by SERS was performed [268]. The preparation of Au NPs dimer was accomplished by linking the NPs with biphenyl-4,4’-dithiol using HOOCPEG-SH as a quenching reagent. Finally, the dimer was modified with the appropriate antibody for the recognition of protein binder used in artworks. The dimer strategy significantly decreased the non-specific aggregation commonly found in immuno-SERS assays. Multiple pathogens were identified by SERS combined with magnetic separation and antibody recognition selection [226]. The advantage of this approach is that lectins, carbohydrate binding proteins that specifically target sugars, were used to modify the magnetic particles, which were then able to recognize the different types of pathogenic bacteria in a sample. Finally, 62

strain-specific antibody-modified SERS-active Au NPs were used for the recognition and detection of these bacteria. SERS was also used for the direct measurement of plant samples. Onion layers were examined by SERS [269]. Chemicals inside the onion layer were analyzed without any requirement for chemical modification or labelling. In addition, when 4-mercaptobenzoic acid was used as SERS probe, the local pH of the onion cell could be examined.

Concluding remarks This review provided an account of some of the developments in the SERS field in the last few years. The amount and the variety of the material covered here illustrate the breadth of the field. Although the focus of this paper is centered in substrate development for applications in analytical/bioanalytical chemistry, the implications of the recent advances in SERS can be felt in other areas of plasmonics and nanotechnology, such as catalysis driven by hot-electron and tip-enhanced Raman scattering, just to name a few. It is clear from this paper that a multitude of SERS substrates with exceptional performance and low cost are already available. The issues of SERS reliability, reproducibility, and quantification at ultra-low concentrations have been tackled using several different approaches, including pre-concentration, digital SERS, and integration of SERS in optical and microfluidic devices. A variety of proof-of-concepts SERS analytical applications have been demonstrated and their performance rivals, and, in most cases, surpass the state-of-the-art based on other techniques, such as fluorescence spectroscopy. Considering all the activity and success of the SERS field, it is somehow surprising that SERS has not yet become a common tool in analytical laboratories. A fact that may be partially responsible for this discrepancy is that SERS applications still require specialists with a multidisciplinary understanding of analytical chemistry, nanotechnology, optics and spectroscopy. This limitation is being overcome by the advent of both commercial high-performance SERS substrates and low cost, miniaturized, Raman spectrometers. Those two factors are transforming SERS into a more accessible tool for non-SERS specialist technical personnel, improving the dissemination of the technique. Moreover, the development of specific SERS-based analytical protocols for niche applications and the utilization of SERS in tandem to other established technical tools, such as capillary 63

electrophoresis or high performance liquid chromatography are becoming more common. All these factors point towards a bright future for SERS in analytical chemistry, where the technique might become the established tool for ultra-sensitive quantification.

Acknowledgements M. Fan would like to thank the financial support from the National Natural Science Foundation of China (no. 21677117). GFSA thanks CNPq for a research fellowship and CNPq and FAPEMIG for financial support. AGB thanks NSERC for continuing support for the research program.

References [1] D.A. Long, The Raman Effect, John Wiley & Sons, Ltd., Chichester, UK., 2001. [2] E.C. Le Ru, P.G. Etchegoin, Principles of Surface-Enhanced Raman Spectroscopy: and related plasmonic effects, Elsevier, Amsterdam, 2009. [3] R. Aroca, Surface-Enhanced Vibrational Spectroscopy, John Wiley & Sons, New York, 2006. [4] M. Moskovits, Surface-enhanced Raman spectroscopy: a brief retrospective, J. Raman Spectrosc., 36 (2005) 485-496. [5] S.-Y. Ding, E.-M. You, Z.-Q. Tian, M. Moskovits, Electromagnetic theories of surfaceenhanced Raman spectroscopy, Chemical Society Reviews, 46 (2017) 4042-4076. [6] M.K. Fan, G.F.S. Andrade, A.G. Brolo, A review on the fabrication of substrates for surface enhanced Raman spectroscopy and their applications in analytical chemistry, Analytica Chimica Acta, 693 (2011) 7-25. [7] J.P. Camden, J.A. Dieringer, J. Zhao, R.P. Van Duyne, Controlled Plasmonic Nanostructures for Surface-Enhanced Spectroscopy and Sensing, Accounts of Chemical Research, 41 (2008) 1653-1661. [8] A. Hakonen, K. Wu, M. Stenbaek Schmidt, P.O. Andersson, A. Boisen, T. Rindzevicius, Detecting forensic substances using commercially available SERS substrates and handheld Raman spectrometers, Talanta, 189 (2018) 649-652. 64

[9] R.A. Alvarez-Puebla, L.M. Liz-Marzán, Traps and cages for universal SERS detection, Chemical Society Reviews, 41 (2012) 43-51. [10] W. Xi, B.K. Shrestha, A.J. Haes, Promoting Intra- and Intermolecular Interactions in Surface-Enhanced Raman Scattering, Analytical Chemistry, 90 (2018) 128-143. [11] J. Reguera, J. Langer, D. Jiménez de Aberasturi, L.M. Liz-Marzán, Anisotropic metal nanoparticles for surface enhanced Raman scattering, Chemical Society Reviews, 46 (2017) 3866-3885. [12] W.B. Li, X.C. Zhao, Z.F. Yi, A.M. Glushenkov, L.X. Kong, Plasmonic substrates for surface enhanced Raman scattering, Analytica Chimica Acta, 984 (2017) 19-41. [13] S. Gwo, C.-Y. Wang, H.-Y. Chen, M.-H. Lin, L. Sun, X. Li, W.-L. Chen, Y.-M. Chang, H. Ahn, Plasmonic Metasurfaces for Nonlinear Optics and Quantitative SERS, ACS Photonics, 3 (2016) 1371-1384. [14] A. Hakonen, P.O. Andersson, M.S. Schmidt, T. Rindzevicius, M. Kall, Explosive and chemical threat detection by surface-enhanced Raman scattering: A review, Analytica Chimica Acta, 893 (2015) 1-13. [15] S. Schlücker, Surface-enhanced Raman spectroscopy: concepts and chemical applications, Angew Chem Int Ed, 53 (2014). [16] S.M. Restaino, I.M. White, A critical review of flexible and porous SERS sensors for analytical chemistry at the point-of-sample, Analytica Chimica Acta, 1060 (2019) 17-29. [17] S. Lee, I. Choi, Fabrication Strategies of 3D Plasmonic Structures for SERS, Biochip Journal, 13 (2019) 30-42. [18] A.H. Nguyen, E.A. Peters, Z.D. Schultz, Bioanalytical applications of surface- enhanced Raman spectroscopy: de novo molecular identification, Reviews in Analytical Chemistry, 36 (2017). [19] K.A. Antonio, Z.D. Schultz, Advances in Biomedical Raman Microscopy, Analytical Chemistry, 86 (2014) 30-46. [20] Y. Liu, H. Zhou, Z. Hu, G. Yu, D. Yang, J. Zhao, Label and label-free based surfaceenhanced Raman scattering for pathogen bacteria detection: A review, Biosensors and Bioelectronics, 94 (2017) 131-140.

65

[21] J.H. Granger, N.E. Schlotter, A.C. Crawford, M.D. Porter, Prospects for point-of-care pathogen diagnostics using surface-enhanced Raman scattering (SERS), Chemical Society Reviews, 45 (2016) 3865-3882. [22] S. Laing, L.E. Jamieson, K. Faulds, D. Graham, Surface-enhanced Raman spectroscopy for in vivo biosensing, Nature Reviews Chemistry, 1 (2017) 0060. [23] J. Kneipp, Interrogating cells, tissues, and live animals with new generations of surfaceenhanced Raman scattering probes and labels, ACS Nano, 11 (2017). [24] L.A. Lane, X. Qian, S. Nie, SERS Nanoparticles in Medicine: From Label-Free Detection to Spectroscopic Tagging, Chemical Reviews, 115 (2015) 10489-10529. [25] S. McAughtrie, K. Faulds, D. Graham, Surface enhanced Raman spectroscopy (SERS): Potential applications for disease detection and treatment, Journal of Photochemistry and Photobiology C: Photochemistry Reviews, 21 (2014) 40-53. [26] L. Rodriguez-Lorenzo, L. Fabris, R.A. Alvarez-Puebla, Multiplex optical sensing with surface-enhanced Raman scattering: A critical review, Analytica Chimica Acta, 745 (2012) 1023. [27] A.-I. Henry, T.W. Ueltschi, M.O. McAnally, R.P. Van Duyne, Spiers Memorial Lecture Surface-enhanced Raman spectroscopy: from single particle/molecule spectroscopy to ångstromscale spatial resolution and femtosecond time resolution, Faraday Discussions, 205 (2017) 9-30. [28] P. Aldeanueva-Potel, E. Carbo-Argibay, N. Pazos-Perez, S. Barbosa, I. Pastoriza-Santos, R.A. Alvarez-Puebla, L.M. Liz-Marzan, Spiked Gold Beads as Substrates for Single-Particle SERS, ChemPhysChem, 13 (2012) 2561-2565. [29] E.C. Le Ru, P.G. Etchegoin, Single-Molecule Surface-Enhanced Raman Spectroscopy, in: M.A. Johnson, T.J. Martinez (Eds.) Annual Review of Physical Chemistry, Vol 632012, pp. 6587. [30] S.-C. Luo, K. Sivashanmugan, J.-D. Liao, C.-K. Yao, H.-C. Peng, Nanofabricated SERSactive substrates for single-molecule to virus detection in vitro: A review, Biosensors and Bioelectronics, 61 (2014) 232-240. [31] D.P. dos Santos, M.L.A. Temperini, A.G. Brolo, Single-Molecule Surface-Enhanced (Resonance) Raman Scattering (SE(R)RS) as a Probe for Metal Colloid Aggregation State, Journal of Physical Chemistry C, 120 (2016) 20877-20885.

66

[32] D.P. dos Santos, M.L.A. Temperini, A.G. Brolo, Intensity Fluctuations in Single-Molecule Surface-Enhanced Raman Scattering, Accounts of Chemical Research, 52 (2019) 456-464. [33] E.C. Le Ru, E. Blackie, M. Meyer, P.G. Etchegoin, Surface enhanced Raman scattering enhancement factors: a comprehensive study, Journal of Physical Chemistry C, 111 (2007) 13794-13803. [34] D.C. Rodrigues, M.L. de Souza, K.S. Souza, D.P. dos Santos, G.F.S. Andrade, M.L.A. Temperini, Critical assessment of enhancement factor measurements in surface-enhanced Raman scattering on different substrates, Physical Chemistry Chemical Physics, 17 (2015) 21294-21301. [35] S. Atta, T.V. Tsoulos, L. Fabris, Shaping Gold Nanostar Electric Fields for SurfaceEnhanced Raman Spectroscopy Enhancement via Silica Coating and Selective Etching, J. Phys. Chem. C, 120 (2016) 20749-20758. [36] C.M.S. Izumi, M.G. Moffitt, A.G. Brolo, Statistics on Surface-Enhanced Resonance Raman Scattering from Single Nanoshells, J. Phys. Chem. C, 115 (2011) 19104-19109. [37] R.W. Taylor, T.C. Lee, O.A. Scherman, R. Esteban, J. Aizpurua, F.M. Huang, J.J. Baumberg, S. Mahajan, Precise Subnanometer Plasmonic Junctions for SERS within Gold Nanoparticle Assemblies Using Cucurbit n uril "Glue", ACS Nano, 5 (2011) 3878-3887. [38] A. Lee, G.F.S. Andrade, A. Ahmed, M.L. Souza, N. Coombs, E. Tumarkin, K. Liu, R. Gordon, A.G. Brolo, E. Kumacheva, Probing Dynamic Generation of Hot-Spots in SelfAssembled Chains of Gold Nanorods by Surface-Enhanced Raman Scattering, J. Am. Chem. Soc., 133 (2011) 7563-7570. [39] J.J. Armao, I. Nyrkova, G. Fuks, A. Osypenko, M. Maaloum, E. Moulin, R. Arenal, O. Gavat, A. Semenov, N. Giuseppone, Anisotropic Self-Assembly of Supramolecular Polymers and Plasmonic Nanoparticles at the Liquid Liquid Interface, J. Am. Chem. Soc., 139 (2017) 2345-2350. [40] A.B. Serrano-Montes, J. Langer, M. Henriksen-Lacey, D.J. de Aberasturi, D.M. Solis, J.M. Taboada, F. Obelleiro, K. Sentosun, S. Bals, A. Bekdemir, F. Stellacci, L.M. Liz-Marzan, Gold Nanostar-Coated Polystyrene Beads as Multifunctional Nanoprobes for SERS Bioimaging, J. Phys. Chem. C, 120 (2016) 20860-20868. [41] N. Cathcart, J.I.L. Chen, V. Kitaev, LSPR Tuning from 470 to 800 nm and Improved Stability of Au-Ag Nanoparticles Formed by Gold Deposition and Rebuilding in the Presence of Poly(styrenesulfonate), Langmuir, 34 (2018) 612-621. 67

[42] J.B. Song, B. Duan, C.X. Wang, J.J. Zhou, L. Pu, Z. Fang, P. Wang, T.T. Lim, H.W. Duan, SERS-Encoded Nanogapped Plasmonic Nanoparticles: Growth of Metallic Nanoshell by Templating Redox-Active Polymer Brushes, J. Am. Chem. Soc., 136 (2014) 6838-6841. [43] P. Liu, H.J. Chen, H. Wang, J.H. Yan, Z.Y. Lin, G.W. Yang, Fabrication of Si/Au Core/Shell Nanoplasmonic Structures with Ultrasensitive Surface-Enhanced Raman Scattering for Mono layer Molecule Detection, J. Phys. Chem. C, 119 (2015) 1234-1246. [44] A.M. Brito-Silva, R.G. Sobral-Filho, R. Barbosa-Silva, C.B. de Araujo, A. Galembeck, A.G. Brolo, Improved synthesis of gold and silver nanoshells, Langmuir, 29 (2013) 4366-4372. [45] T.B.V. Neves, S.M. Landi, L.A. Sena, B.S. Archanjo, G.F.S. Andrade, Silicon dioxide covered Au and Ag nanoparticles for shell-isolated nanoparticle enhanced spectroscopies in the near-infrared, Rsc Advances, 5 (2015) 59373-59378. [46] J.-F. Li, Y.-J. Zhang, S.-Y. Ding, R. Panneerselvam, Z.-Q. Tian, Core–Shell NanoparticleEnhanced Raman Spectroscopy, Chemical Reviews, 117 (2017) 5002-5069. [47] H. Zhang, C. Wang, H.L. Sun, G. Fu, S. Chen, Y.J. Zhang, B.H. Chen, J.R. Anema, Z.L. Yang, J.F. Li, Z.Q. Tian, In situ dynamic tracking of heterogeneous nanocatalytic processes by shell-isolated nanoparticle-enhanced Raman spectroscopy, Nat Commun, 8 (2017) 15447. [48] J.F. Li, Y.F. Huang, Y. Ding, Z.L. Yang, S.B. Li, X.S. Zhou, F.R. Fan, W. Zhang, Z.Y. Zhou, D.Y. Wu, B. Ren, Z.L. Wang, Z.Q. Tian, Shell-isolated nanoparticle-enhanced Raman spectroscopy, Nature, 464 (2010) 392-395. [49] A.S. Indrasekara, S. Meyers, S. Shubeita, L.C. Feldman, T. Gustafsson, L. Fabris, Gold nanostar substrates for SERS-based chemical sensing in the femtomolar regime, Nanoscale, 6 (2014) 8891-8899. [50] H.L. Li, D.D. Men, Y.Q. Sun, D.L. Liu, X.Y. Li, L.B. Li, C.C. Li, W.P. Cai, Y. Li, Surface enhanced Raman scattering properties of dynamically tunable nanogaps between Au nanoparticles self-assembled on hydrogel microspheres controlled by pH, J. Colloid Interface Sci., 505 (2017) 467-475. [51] F. Benz, R. Chikkaraddy, A. Salmon, H. Ohadi, B. de Nijs, J. Mertens, C. Carnegie, R.W. Bowman, J.J. Baumberg, SERS of Individual Nanoparticles on a Mirror: Size Does Matter, but so Does Shape, J. Phys. Chem. Lett., 7 (2016) 2264-2269.

68

[52] J.C. Fraire, V.N.S. Ocello, L.G. Allende, A.V. Veglia, E.A. Coronado, Toward the Design of Highly Stable Small Colloidal SERS Substrates with Supramolecular Host-Guest Interactions for Ultrasensitive Detection, J. Phys. Chem. C, 119 (2015) 8876-8888. [53] C. Carnegie, R. Chikkaraddy, F. Benz, B. de Nijs, W.M. Deacon, M. Horton, W.T. Wang, C. Readman, S.J. Barrow, O.A. Scherman, J.J. Baumberg, Mapping SERS in CB:Au Plasmonic Nanoaggregates, ACS Photonics, 4 (2017) 2681-2686. [54] B. de Nijs, M. Kamp, I. Szabo, S.J. Barrow, F. Benz, G.L. Wu, C. Carnegie, R. Chikkaraddy, W.T. Wang, W.M. Deacon, E. Rosta, J.J. Baumberg, O.A. Scherman, Smart supramolecular sensing with cucurbit n urils: probing hydrogen bonding with SERS, Faraday Discussions, 205 (2017) 505-515. [55] J. Prinz, B. Schreiber, L. Olejko, J. Oertel, J. Rackwitz, A. Keller, I. Bald, DNA Origami Substrates for Highly Sensitive Surface-Enhanced Raman Scattering, J. Phys. Chem. Lett., 4 (2013) 4140-4145. [56] A. Moeinian, F.N. Gur, J. Gonzalez-Torres, L.S. Zhou, V.D. Murugesan, A.D. Dashtestani, H. Guo, T.L. Schmidt, S. Strehle, Highly Localized SERS Measurements Using Single Silicon Nanowires Decorated with DNA Origami-Based SERS Probe, Nano Letters, 19 (2019) 10611066. [57] P.F. Zhan, T. Wen, Z.G. Wang, Y.B. He, J. Shi, T. Wang, X.F. Liu, G.W. Lu, B.Q. Ding, DNA Origami Directed Assembly of Gold Bowtie Nanoantennas for Single-Molecule SurfaceEnhanced Raman Scattering, Angewandte Chemie-International Edition, 57 (2018) 2846-2850. [58] J.H. Lee, J.M. Nam, K.S. Jeon, D.K. Lim, H. Kim, S. Kwon, H. Lee, Y.D. Suh, Tuning and Maximizing the Single-Molecule Surface-Enhanced Raman Scattering from DNA-Tethered Nanodumbbells, ACS Nano, 6 (2012) 9574-9584. [59] J. Prinz, C. Heck, L. Ellerik, V. Merk, I. Bald, DNA origami based Au-Ag-core-shell nanoparticle dimers with single-molecule SERS sensitivity, Nanoscale, 8 (2016) 5612-5620. [60] S. Simoncelli, E.M. Roller, P. Urban, R. Schreiber, A.J. Turberfield, T. Liedl, T. Lohmuller, Quantitative Single-Molecule Surface Enhanced Raman Scattering by Optothermal Tuning of DNA Origami-Assembled Plasmonic Nanoantennas, ACS Nano, 10 (2016) 9809-9815. [61] J. Zheng, Y.P. Hu, J.H. Bai, C. Ma, J.S. Li, Y.H. Li, M.L. Shi, W.H. Tan, R.H. Yang, Universal Surface-Enhanced Raman Scattering Amplification Detector for Ultrasensitive Detection of Multiple Target Analytes, Anal. Chem., 86 (2014) 2205-2212. 69

[62] A.F. Stewart, A. Lee, A. Ahmed, S. Ip, E. Kumacheva, G.C. Walker, Rational Design for the Controlled Aggregation of Gold Nanorods via Phospholipid Encapsulation for Enhanced Raman Scattering, ACS Nano, 8 (2014) 5462-5467. [63] A. Lee, A. Ahmed, D.P. dos Santos, N. Coombs, J.I. Park, R. Gordon, A.G. Brolo, E. Kumacheva, Side-by-Side Assembly of Gold Nanorods Reduces Ensemble-Averaged SERS Intensity, J. Phys. Chem. C, 116 (2012) 5538-5545. [64] Y. Rong, L.P. Song, P. Si, L. Zhang, X.F. Lu, J.W. Zhang, Z.H. Nie, Y.J. Huang, T. Chen, Macroscopic Assembly of Gold Nanorods into Superstructures with Controllable Orientations by Anisotropic Affinity Interaction, Langmuir, 33 (2017) 13867-13873. [65] A. Santinom, M.A. da Silva, J.E.L. Villa, R.J. Poppi, I.O. Mazali, D.P. dos Santos, Surfaceenhanced Raman scattering (SERS) as probe of plasmonic near-field resonances, Vibrational Spectroscopy, 99 (2018) 34-43. [66] L. Polavarapu, L.M. Liz-Marzan, Towards low-cost flexible substrates for nanoplasmonic sensing, Physical Chemistry Chemical Physics, 15 (2013) 5288-5300. [67] A.B.E. Reis, G.P. Oliveira, M.R. Almeida, B. Fragneaud, G.F.S. Andrade, Evaluation of the SERS performance of Ag nanoparticles immobilized on glass by silanes using protic or aprotic solvents, Vibrational Spectroscopy, 87 (2016) 53-59. [68] K. Min, W.J. Jeon, Y. Kim, J.Y. Choi, H.K. Yu, Spontaneous nano-gap formation in Ag film using NaCl sacrificial layer for Raman enhancement, Nanotechnology, 29 (2018). [69] L. Velleman, L. Scarabelli, D. Sikdar, A.A. Kornyshev, L.M. Liz-Marzan, J.B. Edel, Monitoring plasmon coupling and SERS enhancement through in situ nanoparticle spacing modulation, Faraday Discuss., 205 (2017) 67-83. [70] M.P. Konrad, A.P. Doherty, S.E.J. Bell, Stable and Uniform SERS Signals from SelfAssembled Two-Dimensional Interfacial Arrays of Optically Coupled Ag Nanoparticles, Anal. Chem., 85 (2013) 6783-6789. [71] M.P. Cecchini, V.A. Turek, A. Demetriadou, G. Britovsek, T. Welton, A.A. Kornyshev, J.D.E.T. Wilton-Ely, J.B. Edel, Heavy Metal Sensing Using Self-Assembled Nanoparticles at a Liquid-Liquid Interface, Advanced Optical Materials, 2 (2014) 966-977. [72] L. Yan, K. Zhang, H. Xu, J. Ji, Y. Wang, B. Liu, P. Yang, Target induced interfacial selfassembly of nanoparticles: A new platform for reproducible quantification of copper ions, Talanta, 158 (2016) 254-261. 70

[73] L. Zhang, F. Liu, Y. Zou, X. Hu, S. Huang, Y. Xu, L. Zhang, Q. Dong, Z. Liu, L. Chen, Z. Chen, W. Tan, Surfactant-Free Interface Suspended Gold Graphitic Surface-Enhanced Raman Spectroscopy Substrate for Simultaneous Multiphase Analysis, Anal. Chem., 90 (2018) 1118311187. [74] M.P. Cecchini, V.A. Turek, J. Paget, A.A. Kornyshev, J.B. Edel, Self-assembled nanoparticle arrays for multiphase trace analyte detection, Nat Mater, 12 (2013) 165-171. [75] J. Liu, J. Li, F. Li, Y. Zhou, X. Hu, T. Xu, W. Xu, Liquid-liquid interfacial self-assembled Au NP arrays for the rapid and sensitive detection of butyl benzyl phthalate (BBP) by surfaceenhanced Raman spectroscopy, Anal. Bioanal. Chem., 410 (2018) 5277-5285. [76] Y. Ma, H. Liu, M. Mao, J. Meng, L. Yang, J. Liu, Surface-Enhanced Raman Spectroscopy on Liquid Interfacial Nanoparticle Arrays for Multiplex Detecting Drugs in Urine, Anal Chem, 88 (2016) 8145-8151. [77] F. Yu, M. Su, L. Tian, H. Wang, H. Liu, Organic Solvent as Internal Standards for Quantitative and High-Throughput Liquid Interfacial SERS Analysis in Complex Media, Anal. Chem., 90 (2018) 5232-5238. [78] K. Zhang, J. Zhao, J. Ji, Y. Li, B. Liu, Quantitative label-free and real-time surfaceenhanced Raman scattering monitoring of reaction kinetics using self-assembled bifunctional nanoparticle arrays, Anal Chem, 87 (2015) 8702-8708. [79] K. Kim, H.S. Han, I. Choi, C. Lee, S. Hong, S.-H. Suh, L.P. Lee, T. Kang, Interfacial liquidstate surface-enhanced Raman spectroscopy, Nat. Commun., 4 (2013) 2182. [80] M. Mao, B. Zhou, X. Tang, C. Chen, M. Ge, P. Li, X. Huang, L. Yang, J. Liu, Natural Deposition Strategy for Interfacial, Self-Assembled, Large-Scale, Densely Packed, Monolayer Film with Ligand-Exchanged Gold Nanorods for In Situ Surface-Enhanced Raman Scattering Drug Detection, Chemistry-a European Journal, 24 (2018) 4094-4102. [81] S.G. Booth, D.P. Cowcher, R. Goodacre, R.A. Dryfe, Electrochemical modulation of SERS at the liquid/liquid interface, Chem Commun (Camb), 50 (2014) 4482-4484. [82] M. Su, X. Li, S. Zhang, F. Yu, L. Tian, Y. Jiang, H. Liu, Self-Healing Plasmonic Metal Liquid as a Quantitative Surface-Enhanced Raman Scattering Analyzer in Two-Liquid-Phase Systems, Anal. Chem., 91 (2019) 2288-2295. [83] L. Tian, M. Su, F. Yu, Y. Xu, X. Li, L. Li, H. Liu, W. Tan, Liquid-state quantitative SERS analyzer on self-ordered metal liquid-like plasmonic arrays, Nat Commun, 9 (2018) 3642. 71

[84] N. Lebedev, I. Griva, W.J. Dressick, J. Phelps, J.E. Johnson, Y. Meshcheriakova, G.P. Lomonossoff, C.M. Soto, A virus-based nanoplasmonic structure as a surface-enhanced Raman biosensor, Biosens. Bioelectron., 77 (2016) 306-314. [85] C.M. Gabardo, J. Yang, N.J. Smith, R.C. Adams-McGavin, L. Soleymani, Programmable Wrinkling of Self-Assembled Nanoparticle Films on Shape Memory Polymers, ACS Nano, 10 (2016) 8829-8836. [86] Y.H. Lee, C.K. Lee, B.R. Tan, J.M.R. Tan, I.Y. Phang, X.Y. Ling, Using the LangmuirSchaefer technique to fabricate large-area dense SERS-active Au nanoprism monolayer films, Nanoscale, 5 (2013) 6404-6412. [87] J.R. Anema, A.G. Brolo, The use of polarization-dependent SERS from scratched gold films to selectively eliminate solution-phase interference, Plasmonics, 2 (2007) 157-162. [88] G.F.S. Andrade, Q. Min, R. Gordon, A.G. Brolo, Surface-Enhanced Resonance Raman Scattering on Gold Concentric Rings: Polarization Dependence and Intensity Fluctuations, Journal of Physical Chemistry C, 116 (2012) 2672-2676. [89] X.Q. Zhang, W.J. Salcedo, M.M. Rahman, A.G. Brolo, Surface-enhanced Raman scattering from bowtie nanoaperture arrays, Surface Science, 676 (2018) 39-45. [90] A. Ahmed, R. Gordon, Single Molecule Directivity Enhanced Raman Scattering using Nanoantennas, Nano Letters, 12 (2012) 2625-2630. [91] A. Gopalakrishnan, M. Chirumamilla, F. De Angelis, A. Toma, R.P. Zaccaria, R. Krahne, Bimetallic 3D Nanostar Dimers in Ring Cavities: Recyclable and Robust Surface-Enhanced Raman Scattering Substrates for Signal Detection from Few Molecules, Acs Nano, 8 (2014) 7986-7994. [92] F. De Angelis, M. Malerba, M. Patrini, E. Miele, G. Das, A. Toma, R.P. Zaccaria, E. Di Fabrizio, 3D Hollow Nanostructures as Building Blocks for Multifunctional Plasmonics, Nano Letters, 13 (2013) 3553-3558. [93] G.C. Messina, M. Malerba, P. Zilio, E. Miele, M. Dipalo, L. Ferrara, F. De Angelis, Hollow plasmonic antennas for broadband SERS spectroscopy, Beilstein Journal of Nanotechnology, 6 (2015) 492-498. [94] D.C. Rodrigues, G.F. Souza Andrade, M.L. Arruda Temperini, SERS performance of gold nanotubes obtained by sputtering onto polycarbonate track-etched membranes, Physical Chemistry Chemical Physics, 15 (2013) 1169-1176. 72

[95] N.A. Cinel, S. Çakmakyapan, G. Ertaş, E. Özbay, Concentric Ring Structures as Efficient SERS Substrates, IEEE Journal of Selected Topics in Quantum Electronics, 19 (2013) 46016054601605. [96] D.X. Wang, W.Q. Zhu, M.D. Best, J.P. Camden, K.B. Crozier, Directional Raman Scattering from Single Molecules in the Feed Gaps of Optical Antennas, Nano Letters, 13 (2013) 2194-2198. [97] D.X. Wang, W.Q. Zhu, M.D. Best, J.P. Camden, K.B. Crozier, Wafer-scale metasurface for total power absorption, local field enhancement and single molecule Raman spectroscopy, Scientific Reports, 3 (2013). [98] L.L. Dong, X. Yang, C. Zhang, B. Cerjan, L.N. Zhou, M.L. Tseng, Y. Zhang, A. Alabastri, P. Nordlander, N.J. Halas, Nanogapped Au Antennas for Ultrasensitive Surface-Enhanced Infrared Absorption Spectroscopy, Nano Letters, 17 (2017) 5768-5774. [99] M. Tabatabaei, M. Najiminaini, K. Davieau, B. Kaminska, M.R. Singh, J.J.L. Carson, F. Lagugne-Labarthet, Tunable 3D Plasmonic Cavity Nanosensors for Surface-Enhanced Raman Spectroscopy with Sub-femtomolar Limit of Detection, Acs Photonics, 2 (2015) 752-759. [100] M. Manheller, S. Trellenkamp, R. Waser, S. Karthäuser, Reliable fabrication of 3 nm gaps between nanoelectrodes by electron-beam lithography, Nanotechnology, 23 (2012) 125302. [101] J. Zhang, M. Irannejad, B. Cui, Bowtie Nanoantenna with Single-Digit Nanometer Gap for Surface-Enhanced Raman Scattering (SERS), Plasmonics, 10 (2015) 831-837. [102] K. Lee, H. Choi, D.S. Kim, M.S. Jang, M. Choi, Vertical stacking of three-dimensional nanostructures via an aerosol lithography for advanced optical applications, Nanotechnology, 28 (2017). [103] I. Sow, J. Grand, G. Levi, J. Aubard, N. Felidj, J.C. Tinguely, A. Hohenau, J.R. Krenn, Revisiting Surface-Enhanced Raman Scattering on Realistic Lithographic Gold Nanostripes, Journal of Physical Chemistry C, 117 (2013) 25650-25658. [104] V. Merk, J. Kneipp, K. Leosson, Gap Size Reduction and Increased SERS Enhancement in Lithographically Patterned Nanoparticle Arrays by Templated Growth, Advanced Optical Materials, 1 (2013) 313-318. [105] P.J. Kelly, R.D. Arnell, Magnetron sputtering: a review of recent developments and applications, Vacuum, 56 (2000) 159-172.

73

[106] Y. Yang, Z.Y. Li, K. Yamaguchi, M. Tanemura, Z.R. Huang, D.L. Jiang, Y.H. Chen, F. Zhou, M. Nogami, Controlled fabrication of silver nanoneedles array for SERS and their application in rapid detection of narcotics, Nanoscale, 4 (2012) 2663-2669. [107] M. Zhang, A. Zhao, D. Li, H. Sun, D. Wang, H. Guo, Q. Gao, Z. Gan, W. Tao, Generalized green synthesis of diverse LnF3-Ag hybrid architectures and their shape-dependent SERS performances, RSC Advances, 4 (2014) 9205-9212. [108] M. Couture, L.S. Live, A. Dhawan, J.-F. Masson, EOT or Kretschmann configuration? Comparative study of the plasmonic modes in gold nanohole arrays, Analyst, 137 (2012) 41624170. [109] M. Couture, Y.Z. Liang, H.P.P. Richard, R. Faid, W. Peng, J.F. Masson, Tuning the 3D plasmon field of nanohole arrays, Nanoscale, 5 (2013) 12399-12408. [110] H. Im, K.C. Bantz, S.H. Lee, T.W. Johnson, C.L. Haynes, S.H. Oh, Self-Assembled Plasmonic Nanoring Cavity Arrays for SERS and LSPR Biosensing, Advanced Materials, 25 (2013) 2678-2685. [111] G.-N. Xiao, W.-B. Huang, Z.-H. Li, Rapid and Sensitive Detection of Malachite Green and Melamine with Silver Film over Nanospheres by Surface-Enhanced Raman Scattering, Plasmonics, 12 (2017) 1169-1175. [112] X. Dou, P.Y. Chung, H.Y. Sha, Y.C. Lin, P. Jiang, Large-scale fabrication of nanodimple arrays for surface-enhanced Raman scattering, Physical Chemistry Chemical Physics, 15 (2013) 12680-12687. [113] C. Hamon, S. Novikov, L. Scarabelli, L. Basabe-Desmonts, L.M. Liz-Marzan, Hierarchical Self-Assembly of Gold Nanoparticles into Patterned Plasmonic Nanostructures, Acs Nano, 8 (2014) 10694-10703. [114] Y.C. Wang, J.S. DuChene, F.W. Huo, W.D. Wei, An in situ approach for facile fabrication of robust and scalable SERS substrates, Nanoscale, 6 (2014) 7232-7236. [115] C. Hanske, M. Tebbe, C. Kuttner, V. Bieber, V.V. Tsukruk, M. Chanana, T.A.F. Konig, A. Fery, Strongly Coupled Plasmonic Modes on Macroscopic Areas via Template-Assisted Colloidal Self-Assembly, Nano Letters, 14 (2014) 6863-6871. [116] J.W. Jeong, M.M.P. Arnob, K.M. Baek, S.Y. Lee, W.C. Shih, Y.S. Jung, 3D Cross-Point Plasmonic Nanoarchitectures Containing Dense and Regular Hot Spots for Surface-Enhanced Raman Spectroscopy Analysis, Advanced Materials, 28 (2016) 8695-8704. 74

[117] Z. Yi, X. Xu, X. Kang, Y. Zhao, S. Zhang, W. Yao, Y. Yi, J. Luo, C. Wang, Y. Yi, Y. Tang, Fabrication of well-aligned ZnO@Ag nanorod arrays with effective charge transfer for surface-enhanced Raman scattering, Surface and Coatings Technology, 324 (2017) 257-263. [118] Y.-T. Kim, J. Schilling, S.L. Schweizer, R.B. Wehrspohn, High density Ag nanobranches decorated with sputtered Au nanoparticles for surface-enhanced Raman spectroscopy, Applied Surface Science, 410 (2017) 525-529. [119] K. Jiang, J. Wang, Q. Li, L. Liu, C. Liu, S. Fan, Superaligned Carbon Nanotube Arrays, Films, and Yarns: A Road to Applications, Advanced Materials, 23 (2011) 1154-1161. [120] Y.H. Jin, Y.C. Wang, M. Chen, X.Y. Xiao, T.F. Zhang, J.P. Wang, K.L. Jiang, S.S. Fan, Q.Q. Li, Highly Sensitive, Uniform, and Reproducible Surface-Enhanced Raman Spectroscopy Substrate with Nanometer-Scale Quasi-periodic Nanostructures, Acs Applied Materials & Interfaces, 9 (2017) 32369-32376. [121] Q. Zhang, Y.H. Lee, I.Y. Phang, C.K. Lee, X.Y. Ling, Hierarchical 3D SERS Substrates Fabricated by Integrating Photolithographic Microstructures and Self-Assembly of Silver Nanoparticles, Small, 10 (2014) 2703-2711. [122] J.Q. Li, C. Chen, H. Jans, X.M. Xu, N. Verellen, I. Vos, Y. Okumura, V.V. Moshchalkov, L. Lagae, P. Van Dorpe, 300 mm Wafer-level, ultra-dense arrays of Au-capped nanopillars with sub-10 nm gaps as reliable SERS substrates, Nanoscale, 6 (2014) 12391-12396. [123] K.Y. Hong, A.G. Brolo, Polarization-dependent surface-enhanced Raman scattering (SERS) from microarrays, Analytica Chimica Acta, 972 (2017) 73-80. [124] K.Y. Hong, J.W. Menezes, A.G. Brolo, Template-Stripping Fabricated Plasmonic Nanogratings for Chemical Sensing, Plasmonics, 13 (2018) 231-237. [125] J.G. Son, S.W. Han, J.S. Wi, T.G. Lee, Guided formation of sub-5 nm interstitial gaps between plasmonic nanodisks, Nanoscale, 7 (2015) 8338-8342. [126] L. Polavarapu, A. La Porta, S.M. Novikov, M. Coronado-Puchau, L.M. Liz-Marzan, Penon-Paper Approach Toward the Design of Universal Surface Enhanced Raman Scattering Substrates, Small, 10 (2014) 3065-3071. [127] E.P. Hoppmann, W.W. Yu, I.M. White, Highly sensitive and flexible inkjet printed SERS sensors on paper, Methods, 63 (2013) 219-224. [128] A.M. Robinson, L.L. Zhao, M.Y.S. Alam, P. Bhandari, S.G. Harroun, D. Dendukuri, J. Blackburn, C.L. Brosseau, The development of "fab-chips" as low-cost, sensitive surface75

enhanced Raman spectroscopy (SERS) substrates for analytical applications, Analyst, 140 (2015) 779-785. [129] S.Y. Chou, C.C. Yu, Y.T. Yen, K.T. Lin, H.L. Chen, W.F. Su, Romantic Story or Raman Scattering? Rose Petals as Ecofriendly, Low-Cost Substrates for Ultrasensitive SurfaceEnhanced Raman Scattering, Analytical Chemistry, 87 (2015) 6017-6024. [130] B. Sardari, M. Ozcan, Real-Time and Tunable Substrate for Surface Enhanced Raman Spectroscopy by Synthesis of Copper Oxide Nanoparticles via Electrolysis, Sci Rep, 7 (2017) 7730. [131] A. Pallaoro, M.R. Hoonejani, G.B. Braun, C.D. Meinhart, M. Moskovits, Rapid Identification by Surface-Enhanced Raman Spectroscopy of Cancer Cells at Low Concentrations Flowing in a Microfluidic Channel, Acs Nano, 9 (2015) 4328-4336. [132] A. Huefner, W.L. Kuan, K.H. Muller, J.N. Skepper, R.A. Barker, S. Mahajan, Characterization and Visualization of Vesicles in the Endo-Lysosomal Pathway with SurfaceEnhanced Raman Spectroscopy and Chemometrics, Acs Nano, 10 (2016) 307-316. [133] K.Y. Hong, C.D.L. de Albuquerque, R.J. Poppi, A.G. Brolo, Determination of aqueous antibiotic solutions using SERS nanogratings, Anal. Chim. Acta, 982 (2017) 148-155. [134] C.D. Albuquerque, R.J. Poppi, Detection of malathion in food peels by surface-enhanced Raman imaging spectroscopy and multivariate curve resolution, Anal Chim Acta, 879 (2015) 2433. [135] C.P. Shaw, M.K. Fan, C. Lane, G. Barry, A.I. Jirasek, A.G. Brolo, Statistical Correlation Between SERS Intensity and Nanoparticle Cluster Size, J. Phys. Chem. C, 117 (2013) 1659616605. [136] A.C. Crawford, L.B. Laurentius, T.S. Mulvihill, J.H. Granger, J.S. Spencer, D. Chatterjee, K.E. Hanson, M.D. Porter, Detection of the tuberculosis antigenic marker mannose-capped lipoarabinomannan in pretreated serum by surface-enhanced Raman scattering, Analyst, 142 (2017) 186-196. [137] G. Khanderao, J.H. Granger, M.C. Granger, M.A. Firpo, M.D. Porter, S.J. Mulvihill, Optimization Method for the Simultaneous Detection of Multiple Pancreatic Cancer Markers Using a Surface-enhanced Raman Scattering (SERS) Immunoassay, Pancreas, 43 (2014) 13771377.

76

[138] J.H. Granger, M.C. Granger, M.A. Firpo, S.J. Mulvihill, M.D. Porter, Toward development of a surface-enhanced Raman scattering (SERS)-based cancer diagnostic immunoassay panel, Analyst, 138 (2013) 410-416. [139] Y. Xia, K.D. Gilroy, H.-C. Peng, X. Xia, Seed-Mediated Growth of Colloidal Metal Nanocrystals, Angewandte Chemie International Edition, 56 (2017) 60-95. [140] A.X. Wang, X.M. Kong, Review of Recent Progress of Plasmonic Materials and NanoStructures for Surface-Enhanced Raman Scattering, Materials, 8 (2015) 3024-3052. [141] G. Lu, B. Shrestha, A.J. Haes, Importance of Tilt Angles of Adsorbed Aromatic Molecules on Nanoparticle Rattle SERS Substrates, J. Phys. Chem. C, 120 (2016) 20759-20767. [142] B.K. Shrestha, A.J. Haes, Improving surface enhanced Raman signal reproducibility using gold-coated silver nanospheres encapsulated in silica membranes, Journal of Optics, 17 (2015). [143] A.C. Crawford, A. Skuratovsky, M.D. Porter, Sampling Error: Impact on the Quantitative Analysis of Nanoparticle-Based Surface-Enhanced Raman Scattering Immunoassays, Anal. Chem., 88 (2016) 6515-6522. [144] J.T. Hugall, J.J. Baumberg, S. Mahajan, Disentangling the Peak and Background Signals in Surface-Enhanced Raman Scattering, J. Phys. Chem. C, 116 (2012) 6184-6190. [145] Y.F. Tian, P. Wu, Q. Liu, X. Wu, X.D. Hou, Mapping for total surface-enhanced Raman scattering to improve its quantification analysis, Talanta, 161 (2016) 151-156. [146] C. Novara, S. Dalla Marta, A. Virga, A. Lamberti, A. Angelini, A. Chiado, P. Rivolo, F. Geobaldo, V. Sergo, A. Bonifacio, F. Giorgis, SERS-Active Ag Nanoparticles on Porous Silicon and PDMS Substrates: A Comparative Study of Uniformity and Raman Efficiency, J. Phys. Chem. C, 120 (2016) 16946-16953. [147] P.G. Etchegoin, M. Meyer, E.C. Le Ru, Statistics of single molecule SERS signals: is there a Poisson distribution of intensities?, Physical Chemistry Chemical Physics, 9 (2007) 3006-3010. [148] T. Brule, A. Bouhelier, H. Yockell-Lelievre, J.E. Clement, A. Leray, A. Dereux, E. Finot, Statistical and Fourier Analysis for In-line Concentration Sensitivity in Single Molecule Dynamic-SERS, ACS Photonics, 2 (2015) 1266-1271. [149] C.D.L. de Albuquerque, R.G. Sobral-Filho, R.J. Poppi, A.G. Brolo, Digital Protocol for Chemical Analysis at Ultralow Concentrations by Surface-Enhanced Raman Scattering, Anal Chem, 90 (2018) 1248-1254.

77

[150] A. Nguyen, Z.D. Schultz, Quantitative online sheath-flow surface enhanced Raman spectroscopy detection for liquid chromatography, Analyst, 141 (2016) 3630-3635. [151] Y. Zou, L. Chen, Z. Song, D. Ding, Y. Chen, Y. Xu, S. Wang, X. Lai, Y. Zhang, Y. Sun, Z. Chen, W. Tan, Stable and unique graphitic Raman internal standard nanocapsules for surfaceenhanced Raman spectroscopy quantitative analysis, Nano Research, 9 (2016) 1418-1425. [152] Y. Zhang, Y. Zou, F. Liu, Y. Xu, X. Wang, Y. Li, H. Liang, L. Chen, Z. Chen, W. Tan, Stable Graphene-Isolated-Au-Nanocrystal for Accurate and Rapid Surface Enhancement Raman Scattering Analysis, Anal Chem, 88 (2016) 10611-10616. [153] C. Wang, F.S. Cheng, Y.H. Wang, Z.J. Gong, M.K. Fan, J.M. Hu, Single point calibration for semi-quantitative screening based on an internal reference in thin layer chromatographySERS: the case of Rhodamine B in chili oil, Anal. Methods, 6 (2014) 7218-7223. [154] M.K. Fan, P.H. Wang, C. Escobedo, D. Sinton, A.G. Brolo, Surface-enhanced Raman scattering (SERS) optrodes for multiplexed on-chip sensing of nile blue A and oxazine 720, Lab on a Chip, 12 (2012) 1554-1560. [155] S.H. Yazdi, I.M. White, Optofluidic Surface Enhanced Raman Spectroscopy Microsystem for Sensitive and Repeatable On-Site Detection of Chemical Contaminants, Analytical Chemistry, 84 (2012) 7992-7998. [156] J. Jeon, N. Choi, H. Chen, J.I. Moon, L.X. Chen, J. Choo, SERS-based droplet microfluidics for high-throughput gradient analysis, Lab on a Chip, 19 (2019) 674-681. [157] K.K. Reza, A.A. Ibn Sina, A. Wuethrich, Y.S. Grewal, C.B. Howard, D. Korbie, M. Trau, A SERS microfluidic platform for targeting multiple soluble immune checkpoints, Biosensors & Bioelectronics, 126 (2019) 178-186. [158] R.K. Gao, Z.Y. Cheng, X.K. Wang, L.D. Yu, Z.Y. Guo, G. Zhao, J. Choo, Simultaneous immunoassays of dual prostate cancer markers using a SERS-based microdroplet channel, Biosensors & Bioelectronics, 119 (2018) 126-133. [159] B.C. Galarreta, M. Tabatabaei, V. Guieu, E. Peyrin, F. Lagugne-Labarthet, Microfluidic channel with embedded SERS 2D platform for the aptamer detection of ochratoxin A, Analytical and Bioanalytical Chemistry, 405 (2013) 1613-1621. [160] I.J. Hidi, M. Jahn, K. Weber, D. Cialla-May, J. Popp, Droplet based microfluidics: spectroscopic characterization of levofloxacin and its SERS detection, Physical Chemistry Chemical Physics, 17 (2015) 21236-21242. 78

[161] S. Abalde-Cela, C. Abell, R.A. Alvarez-Puebla, L.M. Liz-Marzan, Real Time DualChannel Multiplex SERS Ultradetection, Journal of Physical Chemistry Letters, 5 (2014) 73-79. [162] C. Andreou, M.R. Hoonejani, M.R. Barmi, M. Moskovits, C.D. Meinhart, Rapid Detection of Drugs of Abuse in Saliva Using Surface Enhanced Raman Spectroscopy and Microfluidics, Acs Nano, 7 (2013) 7157-7164. [163] S.H. Yazdi, K.L. Giles, I.M. White, Multiplexed Detection of DNA Sequences Using a Competitive Displacement Assay in a Microfluidic SERRS-Based Device, Analytical Chemistry, 85 (2013) 10605-10611. [164] M.R. Hoonejani, A. Pallaoro, G.B. Braun, M. Moskovits, C.D. Meinhart, Quantitative multiplexed simulated-cell identification by SERS in microfluidic devices, Nanoscale., 7 (2015). [165] A.H. Nguyen, J. Lee, H.I. Choi, H.S. Kwak, S.J. Sim, Fabrication of plasmon length-based surface enhanced Raman scattering for multiplex detection on microfluidic device, Biosensors & Bioelectronics, 70 (2015) 358-365. [166] O. Durucan, T. Rindzevicius, M.S. Schmidt, M. Matteucci, A. Boisen, Nanopillar Filters for Surface-Enhanced Raman Spectroscopy, Acs Sensors, 2 (2017) 1400-1404. [167] M. Li, F.S. Zhao, J.B. Zeng, J. Qi, J. Lu, W.C. Shih, Microfluidic surface-enhanced Raman scattering sensor with monolithically integrated nanoporous gold disk arrays for rapid and labelfree biomolecular detection, Journal of Biomedical Optics, 19 (2014). [168] B.D. Piorek, S.J. Lee, M. Moskovits, C.D. Meinhart, Free-Surface Microfluidics/SurfaceEnhanced Raman Spectroscopy for Real-Time Trace Vapor Detection of Explosives, Analytical Chemistry, 84 (2012) 9700-9705. [169] B.D. Piorek, C. Andreou, M. Moskoyits, C.D. Meinhart, Discrete Free-Surface Millifluidics for Rapid Capture and Analysis of Airborne Molecules Using Surface-Enhanced Raman Spectroscopy, Analytical Chemistry, 86 (2014) 1061-1066. [170] J.W. Martin, M.K. Nieuwoudt, M.J.T. Vargas, O.L.C. Bodley, T.S. Yohendiran, R.N. Oosterbeek, D.E. Williams, M.C. Simpson, Raman on a disc: high-quality Raman spectroscopy in an open channel on a centrifugal microfluidic disc, Analyst, 142 (2017) 1682-1688. [171] S. Cherukulappurath, S.H. Lee, A. Campos, C.L. Haynes, S.H. Oh, Rapid and Sensitive in Situ SERS Detection Using Dielectrophoresis, Chemistry of Materials, 26 (2014) 2445-2452. [172] G.F.S. Andrade, A.G. Brolo, Nanoplasmonic Structures in Optical Fibers, in: A. Dmitriev (Ed.) Nanoplasmonic Sensors, Springer New York, New York, NY, 2012, pp. 289-315. 79

[173] G.F.S. Andrade, J.G. Hayashi, M.M. Rahman, W.J. Salcedo, C.M.B. Cordeiro, A.G. Brolo, Surface-Enhanced Resonance Raman Scattering (SERRS) Using Au Nanohole Arrays on Optical Fiber Tips, Plasmonics, 8 (2013) 1113-1121. [174] F.L. Yap, P. Thoniyot, S. Krishnan, S. Krishnamoorthy, Nanoparticle Cluster Arrays for High-Performance SERS through Directed Self-Assembly on Flat Substrates and on Optical Fibers, Acs Nano, 6 (2012) 2056-2070. [175] M. Pisco, F. Galeotti, G. Quero, G. Grisci, A. Micco, L.V. Mercaldo, P.D. Veneri, A. Cutolo, A. Cusano, Nanosphere lithography for optical fiber tip nanoprobes, Light-Science & Applications, 6 (2017). [176] Y. Liu, Z.L. Huang, F. Zhou, X. Lei, B. Yao, G.W. Meng, Q.H. Mao, Highly sensitive fibre surface-enhanced Raman scattering probes fabricated using laser-induced self-assembly in a meniscus, Nanoscale, 8 (2016) 10607-10614. [177] M.K.K. Oo, Y.B. Guo, K. Reddy, J. Liu, X.D. Fan, Ultrasensitive Vapor Detection with Surface-Enhanced Raman Scattering-Active Gold Nanoparticle Immobilized Flow-Through Multihole Capillaries, Analytical Chemistry, 84 (2012) 3376-3381. [178] H.L. Liu, J. Cao, S. Hanif, C.E. Yuan, J. Pang, R. Levicky, X.H. Xia, K. Wang, SizeControllable Gold Nanopores with High SERS Activity, Analytical Chemistry, 89 (2017) 1040710413. [179] F. De Angelis, F. Gentile, F. Mecarini, G. Das, M. Moretti, P. Candeloro, M.L. Coluccio, G. Cojoc, A. Accardo, C. Liberale, R.P. Zaccaria, G. Perozziello, L. Tirinato, A. Toma, G. Cuda, R. Cingolani, E. Di Fabrizio, Breaking the diffusion limit with super-hydrophobic delivery of molecules to plasmonic nanofocusing SERS structures, Nat Photon, 5 (2011) 682-687. [180] L.-Q. Lu, Y. Zheng, W.-G. Qu, A.-W. Xu, H.-Q. Yu, Hydrophobic Teflon Films as Concentrators for Single-molecule SERS Detection, J. Mater. Chem., 22 (2012) 20986-20990. [181] F. Gentile, M.L. Coluccio, N. Coppedè, F. Mecarini, G. Das, C. Liberale, L. Tirinato, M. Leoncini, G. Perozziello, P. Candeloro, F. De Angelis, E. Di Fabrizio, Superhydrophobic Surfaces as Smart Platforms for the Analysis of Diluted Biological Solutions, ACS Appl. Mater. Interfaces, 4 (2012) 3213-3224. [182] S. Yang, X. Dai, B.B. Stogin, T.-S. Wong, Ultrasensitive surface-enhanced Raman scattering detection in common fluids, Proceedings of the National Academy of Sciences, 113 (2016) 268-273. 80

[183] Z.W. Deng, P.H. Wen, N. Wang, B. Peng, Preparation of hierarchical superhydrophobic melamine-formaldehyde/Ag nanocomposite arrays as surface-enhanced Raman scattering substrates for ultrasensitive and reproducible detection of biomolecules, Sensors and Actuators B-Chemical, 288 (2019) 20-26. [184] Q.Z. Wang, Y.N. Liu, Y.W. Bai, S.Y. Yao, Z.J. Wei, M. Zhang, L.M. Wang, L. Wang, Superhydrophobic SERS substrates based on silver dendrite-decorated filter paper for trace detection of nitenpyram, Analytica Chimica Acta, 1049 (2019) 170-178. [185] J.E. George, V.K. Unnikrishnan, D. Mathur, S. Chidangil, S.D. George, Flexible superhydrophobic SERS substrates fabricated by in situ reduction of Ag on femtosecond laserwritten hierarchical surfaces, Sensors and Actuators B-Chemical, 272 (2018) 485-493. [186] Y.D. Lu, C.J. Wu, R.Y. You, Y. Wu, H.Y. Shen, L.J. Zhu, S.Y. Feng, Superhydrophobic silver film as a SERS substrate for the detection of uric acid and creatinine, Biomedical Optics Express, 9 (2018) 4988-4997. [187] A. Hakonen, T. Rindzevicius, M.S. Schmidt, P.O. Andersson, L. Juhlin, M. Svedendahl, A. Boisen, M. Kall, Detection of nerve gases using surface-enhanced Raman scattering substrates with high droplet adhesion, Nanoscale, 8 (2016) 1305-1308. [188] M.K. Fan, F.S. Cheng, C. Wang, Z.J. Gong, C.Y. Tang, C.Z. Man, A.G. Brolo, SERS optrode as a "fishing rod" to direct preconcentrate analytes from superhydrophobic surfaces, Chem. Commun., 51 (2015) 1965-1968. [189] L. Polavarapu, L.M. Liz-Marzan, Towards low-cost flexible substrates for nanoplasmonic sensing, Phys. Chem. Chem. Phys., 15 (2013) 5288-5300. [190] M. Fan, Z. Zhang, J. Hu, F. Cheng, C. Wang, C. Tang, J. Lin, A.G. Brolo, H. Zhan, Ag decorated sandpaper as flexible SERS substrate for direct swabbing sampling, Mater. Lett., 133 (2014) 57-59. [191] W.W. Yu, I.M. White, Inkjet-printed paper-based SERS dipsticks and swabs for trace chemical detection, Analyst, 138 (2013) 1020-1025. [192] W.W. Yu, I.M. White, Chromatographic separation and detection of target analytes from complex samples using inkjet printed SERS substrates, Analyst, 138 (2013) 3679-3686. [193] W.W. Yu, I.M. White, Inkjet Printed Surface Enhanced Raman Spectroscopy Array on Cellulose Paper, Anal. Chem., 82 (2010) 9626-9630.

81

[194] A. Martín, J.J. Wang, D. Iacopino, Flexible SERS active substrates from ordered vertical Au nanorod arrays, RSC Advances, 4 (2014) 20038. [195] C.H. Lee, L. Tian, S. Singamaneni, Paper-based SERS swab for rapid trace detection on real-world surfaces, ACS applied materials & interfaces, 2 (2010) 3429-3435. [196] C.H. Lee, M.E. Hankus, L.M. Tian, P.M. Pellegrino, S. Singamaneni, Plasmonic paper as a highly efficient SERS substrate, in: A.W. Fountain (Ed.) Chemical, Biological, Radiological, Nuclear, and Explosives2012. [197] L.B. Zhong, J. Yin, Y.M. Zheng, Q. Liu, X.X. Cheng, F.H. Luo, Self-assembly of Au nanoparticles on PMMA template as flexible, transparent, and highly active SERS substrates, Anal. Chem., 86 (2014) 6262-6267. [198] K.J. Si, P. Guo, Q. Shi, W. Cheng, Self-Assembled Nanocube-Based Plasmene Nanosheets as Soft Surface-Enhanced Raman Scattering Substrates toward Direct Quantitative Drug Identification on Surfaces, Analytical Chemistry, 87 (2015) 5263-5269. [199] J.R. Verkouteren, J.L. Coleman, R.A. Fletcher, W.J. Smith, G.A. Klouda, G. Gillen, A method to determine collection efficiency of particles by swipe sampling, Meas. Sci. Technol., 19 (2008) 115101. [200] Y.S. Sui, G.H. Wan, Y.W. Chen, H.L. Ku, L.P. Li, C.H. Liu, H.S. Mau, Effectiveness of Bacterial Disinfectants on Surfaces of Mechanical Ventilator Systems, Respiratory Care, 57 (2012) 250-256. [201] U. Rabuza, S. Sostar-Turk, S. Fijan, Efficiency of four sampling methods used to detect two common nosocomial pathogens on textiles, Text. Res. J., 82 (2012) 2099-2105. [202] J.L. Staymates, J. Grandner, G. Gillen, Fabrication of adhesive coated swabs for improved swipe-based particle collection efficiency, Analytical Methods, 3 (2011) 2056-2060. [203] Z. Gong, H. Du, F. Cheng, C. Wang, C. Wang, M. Fan, Fabrication of SERS swab for direct detection of trace explosives in fingerprints, ACS applied materials & interfaces, 6 (2014) 21931-21937. [204] M. Leona, P. Decuzzi, T.A. Kubic, G. Gates, J.R. Lombardi, Nondestructive identification of natural and synthetic organic colorants in works of art by surface enhanced Raman scattering, Analytical chemistry, 83 (2011) 3990-3993. [205] E. Platania, J.R. Lombardi, M. Leona, N. Shibayama, C. Lofrumento, M. Ricci, M. Becucci, E. Castellucci, Suitability of Ag‐agar gel for the micro‐extraction of organic dyes on 82

different substrates: the case study of wool, silk, printed cotton and a panel painting mock‐up, Journal of Raman Spectroscopy, 45 (2014) 1133-1139. [206] C. Lofrumento, M. Ricci, E. Platania, M. Becucci, E. Castellucci, SERS detection of red organic dyes in Ag-agar gel, J. Raman Spectrosc., 44 (2013) 47-54. [207] A. Raza, B. Saha, Silver nanoparticles doped agarose disk: Highly sensitive surfaceenhanced Raman scattering substrate for in situ analysis of ink dyes, Forensic Science International, 233 (2013) 21-27. [208] P. Aldeanueva-Potel, E. Faoucher, R.n.A. Alvarez-Puebla, L.M. Liz-Marzán, M. Brust, Recyclable molecular trapping and SERS detection in silver-loaded agarose gels with dynamic hot spots, Analytical chemistry, 81 (2009) 9233-9238. [209] Z. Huang, G. Meng, Q. Huang, B. Chen, F. Zhou, X. Hu, Y. Qian, H. Tang, F. Han, Z. Chu, Polyacrylic acid sodium salt film entrapped Ag-nanocubes as molecule traps for SERS detection, Nano Research, 7 (2014) 1177-1187. [210] K. Shin, K. Ryu, H. Lee, K. Kim, H. Chung, D. Sohn, Au nanoparticle-encapsulated hydrogel substrates for robust and reproducible SERS measurement, Analyst, 138 (2013) 932938. [211] Z. Gong, C. Wang, C. Wang, C. Tang, F. Cheng, H. Du, M. Fan, A.G. Brolo, A silver nanoparticle embedded hydrogel as a substrate for surface contamination analysis by surfaceenhanced Raman scattering, Analyst, 139 (2014) 5283-5289. [212] C. Wang, K.W. Wong, Q. Wang, Y. Zhou, C. Tang, M. Fan, J. Mei, W.-M. Lau, Silvernanoparticles-loaded chitosan foam as a flexible SERS substrate for active collecting analytes from both solid surface and solution, Talanta, 191 (2019) 241-247. [213] J. Sun, L. Gong, Y. Lu, D. Wang, Z. Gong, M. Fan, Dual functional PDMS sponge SERS substrate for the on-site detection of pesticides both on fruit surfaces and in juice, Analyst, 143 (2018) 2689-2695. [214] J.B. Song, J.J. Zhou, H.W. Duan, Self-Assembled Plasmonic Vesicles of SERS-Encoded Amphiphilic Gold Nanoparticles for Cancer Cell Targeting and Traceable Intracellular Drug Delivery, Journal of the American Chemical Society, 134 (2012) 13458-13469. [215] C.Y. Hu, J.L. Shen, J. Yan, J. Zhong, W.W. Qin, R. Liu, A. Aldalbahi, X.L. Zuo, S.P. Song, C.H. Fan, D.N. He, Highly narrow nanogap-containing Au@Au core-shell SERS

83

nanoparticles: size-dependent Raman enhancement and applications in cancer cell imaging, Nanoscale, 8 (2016) 2090-2096. [216] C.M. MacLaughlin, N. Mullaithilaga, G.S. Yang, S.Y. Ip, C. Wang, G.C. Walker, SurfaceEnhanced Raman Scattering Dye-Labeled Au Nanoparticles for Triplexed Detection of Leukemia and Lymphoma Cells and SERS Flow Cytometry, Langmuir, 29 (2013) 1908-1919. [217] C.Y. Liu, Y.Y. Han, P.H. Shih, W.N. Lian, H.H. Wang, C.H. Lin, P.R. Hsueh, J.K. Wang, Y.L. Wang, Rapid bacterial antibiotic susceptibility test based on simple surface-enhanced Raman spectroscopic biomarkers, Scientific Reports, 6 (2016). [218] D. Craig, S. McAughtrie, J. Simpson, C. McCraw, K. Faulds, D. Graham, Confocal SERS Mapping of Glycan Expression for the Identification of Cancerous Cells, Analytical Chemistry, 86 (2014) 4775-4782. [219] D. Drescher, T. Buchner, D. McNaughton, J. Kneipp, SERS reveals the specific interaction of silver and gold nanoparticles with hemoglobin and red blood cell components, Physical Chemistry Chemical Physics, 15 (2013) 5364-5373. [220] S.A. Camacho, R.G. Sobral, P.H.B. Aoki, C.J.L. Constantino, A.G. Brolo, Immunoassay quantification using surface-enhanced fluorescence (SEF) tags, Analyst, 142 (2017) 2717-2724. [221] S.A. Camacho, R.G. Sobral-Filho, P.H.B. Aoki, C.J.L. Constantino, A.G. Brolo, Zika Immunoassay Based on Surface-Enhanced Raman Scattering Nanoprobes, ACS Sens., 3 (2018) 587-594. [222] S.L. Filbrun, A.B. Filbrun, F.L. Lovato, S.H. Oh, E.A. Driskell, J.D. Driskell, Chemical modification of antibodies enables the formation of stable antibody-gold nanoparticle conjugates for biosensing, Analyst, 142 (2017) 4456-4467. [223] A. Lopez, F. Lovato, S.H. Oh, Y.H. Lai, S. Filbrun, E.A. Driskell, J.D. Driskell, SERS immunoassay based on the capture and concentration of antigen-assembled gold nanoparticles, Talanta, 146 (2016) 388-393. [224] L.B. Laurentius, A.C. Crawford, T.S. Mulvihill, J.H. Granger, R. Robinson, J.S. Spencer, D. Chatterjee, K.E. Hanson, M.D. Porter, Importance of specimen pretreatment for the low-level detection of mycobacterial lipoarabinomannan in human serum, Analyst, 142 (2017) 177-185. [225] Y. Ma, K. Promthaveepong, N. Li, Highly Bright SERS Nanotags with Multiplexing Fingerprints for Sensitive Immunoassays, Advanced Optical Materials, 5 (2017).

84

[226] H. Kearns, R. Goodacre, L.E. Jamieson, D. Graham, K. Faulds, SERS Detection of Multiple Antimicrobial-Resistant Pathogens Using Nanosensors, Anal Chem, 89 (2017) 1266612673. [227] O.J.R. Clarke, B.L. Goodall, H.P. Hui, N. Vats, C.L. Brosseau, Development of a SERSBased Rapid Vertical Flow Assay for Point-of Care Diagnostics, Analytical Chemistry, 89 (2017) 1405-1410. [228] L. Guerrini, Z. Krpetic, D. van Lierop, R.A. Alvarez-Puebla, D. Graham, Direct SurfaceEnhanced Raman Scattering Analysis of DNA Duplexes, Angew. Chem.-Int. Edit., 54 (2015) 1144-1148. [229] L.-J. Xu, Z.-C. Lei, J. Li, C. Zong, C.J. Yang, B. Ren, Label-Free Surface-Enhanced Raman Spectroscopy Detection of DNA with Single-Base Sensitivity, J. Am. Chem. Soc., 137 (2015) 5149-5154. [230] K. Gracie, E. Correa, S. Mabbott, J.A. Dougan, D. Graham, R. Goodacre, K. Faulds, Simultaneous detection and quantification of three bacterial meningitis pathogens by SERS, Chem. Sci., 5 (2014) 1030-1040. [231] U.S. Kadam, R.L. Chavhan, B. Schulz, J. Irudayaraj, Single molecule Raman spectroscopic assay to detect transgene from GM plants, Analytical Biochemistry, 532 (2017) 60-63. [232] H.N. Xie, Y.Y. Lin, M. Mazo, C. Chiappini, A. Sanchez-Iglesias, L.M. Liz-Marzan, M.M. Stevens, Identification of intracellular gold nanoparticles using surface-enhanced Raman scattering, Nanoscale, 6 (2014) 12403-12407. [233] F. Lussier, T. Brule, M. Vishwakarma, T. Das, J.P. Spatz, J.F. Masson, Dynamic-SERS Optophysiology: A Nanosensor for Monitoring Cell Secretion Events, Nano Letters, 16 (2016) 3866-3871. [234] F. Lussier, T. Brule, M.J. Bourque, C. Ducrot, L.E. Trudeau, J.F. Masson, Dynamic SERS nanosensor for neurotransmitter sensing near neurons, Faraday Discussions, 205 (2017) 387-407. [235] D. Bhowmik, K.R. Mote, C.M. MacLaughlin, N. Biswas, B. Chandra, J.K. Basu, G.C. Walker, P.K. Madhu, S. Maiti, Cell-Membrane-Mimicking Lipid-Coated Nanoparticles Confer Raman Enhancement to Membrane Proteins and Reveal Membrane-Attached Amyloid-beta Conformation, Acs Nano, 9 (2015) 9070-9077.

85

[236] C.H. Zhang, L.W. Liu, P. Liang, L.J. Tang, R.Q. Yu, J.H. Jiang, Plasmon Coupling Enhanced Raman Scattering Nanobeacon for Single-Step, Ultrasensitive Detection of Cholera Toxin, Analytical Chemistry, 88 (2016) 7447-7452. [237] C. Costas, V. Lopez-Puente, G. Bodelon, C. Gonzalez-Bello, J. Perez-Juste, I. PastorizaSantos, L.M. Liz-Marzan, Using Surface Enhanced Raman Scattering to Analyze the Interactions of Protein Receptors with Bacterial Quorum Sensing Modulators, Acs Nano, 9 (2015) 55675576. [238] G. Bodelon, V. Montes-Garcia, V. Lopez-Puente, E.H. Hill, C. Hamon, M.N. Sanz-Ortiz, S. Rodal-Cedeira, C. Costas, S. Celiksoy, I. Perez-Juste, L. Scarabelli, A. La Porta, J. PerezJuste, I. Pastoriza-Santos, L.M. Liz-Marzan, Detection and imaging of quorum sensing in Pseudomonas aeruginosa biofilm communities by surface-enhanced resonance Raman scattering, Nature Materials, 15 (2016) 1203-+. [239] X. Gu, H. Wang, Z.D. Schultz, J.P. Camden, Sensing Glucose in Urine and Serum and Hydrogen Peroxide in Living Cells by Use of a Novel Boronate Nanoprobe Based on SurfaceEnhanced Raman Spectroscopy, Analytical Chemistry, 88 (2016) 7191-7197. [240] Z. Zhang, W. Yu, J. Wang, D. Luo, X.Z. Qiao, X.Y. Qin, T. Wang, Ultrasensitive SurfaceEnhanced Raman Scattering Sensor of Gaseous Aldehydes as Biomarkers of Lung Cancer on Dendritic Ag Nanocrystals, Analytical Chemistry, 89 (2017) 1416-1420. [241] S.M. Asiala, N.C. Shand, K. Faulds, D. Graham, Surface-Enhanced, Spatially Offset Raman Spectroscopy (SESORS) in Tissue Analogues, Acs Applied Materials & Interfaces, 9 (2017) 25488-25494. [242] M. Lopez-Lopez, V. Merk, C. Garcia-Ruiz, J. Kneipp, Surface-enhanced Raman spectroscopy for the analysis of smokeless gunpowders and macroscopic gunshot residues, Anal. Bioanal. Chem., 408 (2016) 4965-4973. [243] F.S. Cheng, H.B. Xu, C. Wang, Z.J. Gong, C.Y. Tang, M.K. Fan, Surface enhanced Raman scattering fiber optic sensor as an ion selective optrode: the example of Cd2+ detection, RSC Adv., 4 (2014) 64683-64687. [244] J. Docherty, S. Mabbott, E. Smith, K. Faulds, C. Davidson, J. Reglinski, D. Graham, Detection of potentially toxic metals by SERS using salen complexes, Analyst, 141 (2016) 58575863.

86

[245] Y. Shi, H. Wang, X. Jiang, B. Sun, B. Song, Y. Su, Y. He, Ultrasensitive, Specific, Recyclable, and Reproducible Detection of Lead Ions in Real Systems through a PolyadenineAssisted, Surface-Enhanced Raman Scattering Silicon Chip, Anal. Chem., 88 (2016) 3723-3729. [246] G. Lu, T.Z. Forbes, A.J. Haes, SERS detection of uranyl using functionalized gold nanostars promoted by nanoparticle shape and size, Analyst, 141 (2016) 5137-5143. [247] H.K. Choi, W.H. Park, C.G. Park, H.H. Shin, K.S. Lee, Z.H. Kirn, Metal-Catalyzed Chemical Reaction of Single Molecules Directly Probed by Vibrational Spectroscopy, J. Am. Chem. Soc., 138 (2016) 4673-4684. [248] G.C. Zheng, L. Polavarapu, L.M. Liz-Marzan, I. Pastoriza-Santos, J. Perez-Juste, Gold nanoparticle-loaded filter paper: a recyclable dip-catalyst for real-time reaction monitoring by surface enhanced Raman scattering, Chem. Commun., 51 (2015) 4572-4575. [249] M. Lee, K. Oh, H.K. Choi, S.G. Lee, H.J. Youn, H.L. Lee, D.H. Jeong, Subnanomolar Sensitivity of Filter Paper-Based SERS Sensor for Pesticide Detection by Hydrophobicity Change of Paper Surface, ACS Sens., 3 (2018) 151-159. [250] C. Zhang, T. You, N. Yang, Y. Gao, L. Jiang, P. Yin, Hydrophobic paper-based SERS platform for direct-droplet quantitative determination of melamine, Food Chemistry, 287 (2019) 363-368. [251] P. Reokrungruang, I. Chatnuntawech, T. Dharakul, S. Bamrungsap, A simple paper-based surface enhanced Raman scattering (SERS) platform and magnetic separation for cancer screening, Sensors and Actuators, B: Chemical, (2019) 462-469. [252] F. Zeng, T. Mou, C. Zhang, X. Huang, B. Wang, X. Ma, J. Guo, Paper-based SERS analysis with smartphones as Raman spectral analyzers, Analyst, 144 (2019) 137-142. [253] S. Patze, U. Huebner, F. Liebold, K. Weber, D. Cialla-May, J. Popp, SERS as an analytical tool in environmental science: The detection of sulfamethoxazole in the nanomolar range by applying a microfluidic cartridge setup, Anal. Chim. Acta, 949 (2017) 1-7. [254] K.S. McKeating, M. Couture, M.P. Dinel, S. Garneau-Tsodikova, J.F. Masson, High throughput LSPR and SERS analysis of aminoglycoside antibiotics, Analyst, 141 (2016) 51205126. [255] S. Ben-Jaber, W.J. Peveler, R. Quesada-Cabrera, E. Cortes, C. Sotelo-Vazquez, N. AbdulKarim, S.A. Maier, I.P. Parkin, Photo-induced enhanced Raman spectroscopy for universal ultratrace detection of explosives, pollutants and biomolecules, Nat. Commun., 7 (2016). 87

[256] S. Ben-Jaber, W.J. Peveler, R. Quesada-Cabrera, C.W.O. Sol, I. Papakonstantinou, I.P. Parkin, Sensitive and specific detection of explosives in solution and vapour by surfaceenhanced Raman spectroscopy on silver nanocubes, Nanoscale, 9 (2017) 16459-16466. [257] B.H.C. Greene, D.S. Alhatab, C.C. Pye, C.L. Brosseau, Electrochemical-Surface Enhanced Raman Spectroscopic (EC-SERS) Study of 6-Thiouric Acid: A Metabolite of the Chemotherapy Drug Azathioprine, J. Phys. Chem. C, 121 (2017) 8084-8090. [258] M.R. Bailey, R.S. Martin, Z.D. Schultz, Role of Surface Adsorption in the SurfaceEnhanced Raman Scattering and Electrochemical Detection of Neurotransmitters, J. Phys. Chem. C, 120 (2016) 20624-20633. [259] A.L. Filgueiras, F.R.A. Lima, D.F. de Carvalho, M.A. Meirelles, D. Paschoal, H.F. dos Santos, S. Sanchez-Cortes, A.C. Sant'Ana, The adsorption of rifampicin on gold or silver surfaces mediated by 2-mercaptoethanol investigated by surface-enhanced Raman scattering spectroscopy, Vibrational Spectroscopy, 86 (2016) 75-80. [260] L.D. Noman, A.C. Sant'Ana, The control of the adsorption of bovine serum albumin on mercaptan-modified gold thin films investigated by SERS spectroscopy, Spectrochimica Acta Part a-Molecular and Biomolecular Spectroscopy, 204 (2018) 119-124. [261] L. Zhang, J.J. Xu, L. Mi, H. Gong, S.Y. Jiang, Q.M. Yu, Multifunctional magneticplasmonic nanoparticles for fast concentration and sensitive detection of bacteria using SERS, Biosensors & Bioelectronics, 31 (2012) 130-136. [262] S.H. Toma, J.J. Santos, K. Araki, H.E. Toma, Pushing the surface-enhanced Raman scattering analyses sensitivity by magnetic concentration: A simple non core-shell approach, Anal. Chim. Acta, 855 (2015) 70-75. [263] H. Qu, Y.M. Lai, D.Z. Niu, S.Q. Sun, Surface-enhanced Raman scattering from magnetometal nanoparticle assemblies, Analytica Chimica Acta, 763 (2013) 38-42. [264] P. Zhao, H.X. Li, D.W. Li, Y.J. Hou, L. Mao, M. Yang, Y. Wang, A SERS nano-tag-based magnetic-separation strategy for highly sensitive immunoassay in unprocessed whole blood, Talanta, 198 (2019) 527-533. [265] P. Negri, K.T. Jacobs, O.O. Dada, Z.D. Schultz, Ultrasensitive Surface-Enhanced Raman Scattering Flow Detector Using Hydrodynamic Focusing, Anal. Chem., 85 (2013) 10159-10166. [266] P. Negri, Z.D. Schultz, Online SERS detection of the 20 proteinogenic L-amino acids separated by capillary zone electrophoresis, Analyst, 139 (2014) 5989-5998. 88

[267] P. Negri, S.A. Sarver, N.M. Schiavone, N.J. Dovichi, Z.D. Schultz, Online SERS detection and characterization of eight biologically-active peptides separated by capillary zone electrophoresis, Analyst, 140 (2015) 1516-1522. [268] E.A. Perets, A. Indrasekara, A. Kurmis, N. Atlasevich, L. Fabris, J. Arslanoglu, Carboxyterminated immuno-SERS tags overcome non-specific aggregation for the robust detection and localization of organic media in artworks, Analyst, 140 (2015) 5971-5980. [269] M.E. Palanco, K.B. Mogensen, K. Kneipp, Raman spectroscopic probing of plant material using SERS, J. Raman Spectrosc., 47 (2016) 156-161.

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Figure Captions

Figure 1. Selected examples of controlled shape, size distribution and aggregation state in colloidal systems. A) cucumber alike gold nanostars (NSt), adapted from ref. [28] © 2012 WileyVCH, used with permission; b) silica coated Au NSt, adapted from ref. [35]. © 2016 American Chemical Society; used with permission; c) Histogram of integrated SERRS intensities normalized by the average of individual nanoshells (N = 177)[36]. © 2011 American Chemical Society; used with permission; d) AuNPs dimerized by CB[n] with portal-to-portal separation rigidly fixed at 0.9 nm, adapted from ref. [37]. © 2011 American Chemical Society; used with permission; e) SERS peak intensity measured at 563 cm-1 and the product of extinction plotted as a function of the average aggregation number of the NR chains, adapted from ref. [38]. © 2011 American Chemical Society; used with permission; f) TEM images of the Au NPs film templated using supramolecular polymer, adapted from ref. [39]. © 2017 American Chemical Society; used with permission.

Figure 2. Left, variation of normalized SERS intensity of assembled Au NR with time. Right, electric field intensity profiles produced via 3D-FDTD simulation for side-by-side assembled NR from Ref [63]. ©2012 American Chemical Society; used with permission.

Figure 3. A multiphase liquid-state SERS analyzer. Reversible O/W encasing for self-assembly of metal liquid-like GNR arrays is realized in a common cuvette[83]. © 2018 Nature; used with permission.

Figure 4. (A) Optical microscopy of a concentric rings structure milled on a 300 nm thick gold film. The ring periodicity is 870 nm, and the strip width is 435 nm. The scale bar is 20 µm. (B) Scanning electron microscopy image of the rings. The scale bar is 20 µm. 2D-mapping of the SERS intensity of the 594 cm-1 band for 1 µmol L-1 NBA on the Au concentric rings structure 90

for the laser light polarized (C) in the y direction and (D) in the x direction, as indicated in panel A. The spectra were obtained at each 1 µm in both x and y directions. Reproduced from ref. [88] © 2012 American Chemical Society; used with permission.

Figure 5. (a) SEM image of the large area nanograting substrate. (b) White light diffraction picture of the gold-coated µ-arrays. The inset is the SEM image of one of the µ-arrays. The blue scale bar is 1 mm. (c) The average SERS spectra of 4-mercaptopyridine (adsorbed from 10 mM solution) (red) TM/perpendicular polarization and (blue) TE/parallel polarization against the nanogratings main directions. Polarization directions are illustrated in the figure. Excitation: 633 nm. Adapted from ref. [123] © 2017 Elsevier B.V. Used with permission.

Figure 6. (a) Plot of the average values of the non-negative matrix factorization with alternating least-squares algorithm (NMF-ALS) at 95% confidence level versus surface density of the analyte (enrofloxacin); (b) Calibration curve build with digitalized SERS mapping for enrofloxacin at ultralow concentration. [149] ©2018 American Chemical Society, used with permission.

Figure 7. a) Optofluidic SERS microsystem with packed microspheres for passive concentration, an integrated micromixer to promote adsorption of the target analyte, and integrated fiber optic cables for optical excitation and collection[155]. ©2017 American Chemical Society, used with permission.; b) Photographs of fabricated centrifugal microfluidic SERS platform, adapted from ref. [166]. ©2017 American Chemical Society, used with permission.; c) Cutaway illustration of material flows in the free-surface microfluidic channel. The aqueous microfluidic phase flows from left to right (blue arrows). The gas phase flows from back to front (green arrows). Analyte molecules (red spheres) diffuse from the gas phase into the liquid phase (red arrows). Nanoparticles (white spheres) suspended in the aqueous phase adsorb to suspended analyte molecules before interrogation by 658 nm laser light (red vertical beam) for detection by SERS[167]. ©2014 SPIE, used with permission; d) Centrifugal microfluidic disc with open 91

Raman spectroscopy measurement chambers [170]. ©2017 Royal Society of Chemistry, used with permission..

Figure 8. (a) Schematic representation of the microfluidic gradient chip. Two different analyte solutions at the same concentration were simultaneously pumped into the microfluidic chip through inlets 1 and 2. At the straight portion of each channel (shown by arrows), small holes were punched with a syringe needle and the SERS optrodes were introduced. The other ends of the optrodes were linked to a Raman microscope; (b) labeled picture of the experimental setup for on-chip multiplex detection; and (c) schematic of the fiber optrode tip sensor with nanoparticles and linker. The core and clad size of the fiber used in this report are 62.5 mm and 125 mm respectively. (d) In-line monitoring of the flow of analytes: (i) R6G (10 µM), (ii) CG (5 µM), (iii) oxa (5 µM), (iv) NBA (5 µM), and (v) CB (10 µM). All spectra are background

corrected and offset for clarity. The numbers on the spectra indicate the main vibrational features (in cm-1) from the respective dye. The x-coordinate (Raman shift) for the spectra i, ii, and v is at the bottom of the graph; and the x-coordinate (Raman shift) for the spectra iii and iv is at the top of the graph. The solutions were introduced into the channel sequentially from i to v (from bottom to top, as shown by the arrow). Note that R6G and CG are not in resonance with the laser and their spectra are due to SERS, but the other three dyes are also in resonance (SERRS effect). (e) Average spectra from each channel of the dilution chip; inset shows the enlarged portion of the spectral region where bands from each dye in the mixture can be uniquely identified.

At

682

cm-1 (inset), top to bottom, spectra are from outlet channel 1 to channel 5, respectively. (f) SERRS fractions of both dyes plotted against their respective concentrations in each channel. The error bars show the variation over three measurements in the same channel. Adapted from [154]. © 2012, The Royal Society of Chemistry. Used with permission.

Figure 9. (A) Swabbing test on a whole pear. The inset shows SEM of the SERS swab; (B) SERS spectra of triazophos obtained by swabbing different surfaces. (a) Pear (~0.3cm2, 53.3 pmol cm2); (b) tree leaf (~0.6cm2, 266 pmol cm-2); (c) plastic (~3cm2, 10.5 pmol cm-2); (d) glass

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(~4cm2, 4.2 pmol cm-2); (e) 5uL 1ppm triazophos. The arrows show the most dominant bands of triazophos. © 2014 Elsevier, used with permission.

Figure 10. The preparation of the slime SERS substrate and SERS of the surface contaminant with the shape adaptable substrate,(a) AgNP–PVA mixture; (b) sodium borate; (c) SERS slime; (d) wood stick; (e) SERS slime on a wood stick; (f) contaminated surface; (g), SERS of the surface contaminant [211]. © 2014 Royal Society of Chemistry, used with permission.

Figure 11. Schematic illustration of (a) Zika-mAb-SERS nanoprobe assembly: Au-SHIN (∼100 nm Au core + 4 nm silica shell thickness); Au-SHIN + NBARaman reporter layer; Au-SHIN + NB Raman reporter layer + final ∼10 nm silica shell (SERS nanoprobe); conjugation onto Zika NS1 monoclonal antibodies (Zika-mAb). (b) SERS immunoassay platform for detecting different concentrations of Zika NS1. The platform is irradiated with 632.8 nm laser line and the SERRS signal from NBA molecules, located in a close distance of gold nanoparticles (∼4 nm), are recorded by area mappings. Brighter spots indicate higher intensity of the NB band at 593 cm−1. Adapted

from ref. [221].© 2018 American Chemical Society; used with permission.

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A Review on Recent Advances in the Applications of SurfaceEnhanced Raman Scattering in Analytical Chemistry Meikun Fan, Gustavo F. S. Andrade, Alexandre G. Brolo

Highlights:

• • • • •

This review covers recent advances of SERS applications in analytical chemistry Progresses in substrate development are discussed. New types of devices that use SERS as a detection platform have been introduced. SERS for bioanalytical applications is the area with most activity in the field. New SERS quantification techniques are now available.

Author’s Biography

Dr. Gustavo F. S. Andrade "Dr. Gustavo F. S. Andrade obtained his Bachelor, Master, and Doctoral degrees from the Institute of Chemistry of the University of Sao Paulo in Brazil in 2007. In 2008-2009 he was awarded a postdoctoral fellow in the University of Victoria (Canada) working with Dr. Alexandre Brolo. He is now an associate professor at the Department of Chemistry of the University of Juiz de Fora in Brazil. His research interests are on surface-enhanced Raman scattering (SERS) and plasmonics and their applications in biosensing and environmental remediation."

Author’s Biography

Dr. Alexandre G. Brolo

“Alexandre G. Brolo is Professor of Chemistry at the University of Victoria in British Columbia, Canada. He obtained his M.Sc. from the University of Sao Paulo (Brazil) and his Ph.D. from the University of Waterloo (Canada). Dr. Brolo’s research interest are of the fabrication of nanostructured metal surfaces; the investigation of their optical properties; and their application in analytical chemistry. He is well-known for his work on the development of new types of surface Plasmon resonance sensors and on the field of surface-enhanced spectroscopy, particularly on surface-enhanced Raman scattering (SERS).”

Author’s Biography Dr. Meikun Fan

"Dr. Meikun Fan obtained his Bachelor’s (1999) and Master’s degree (2002) from Southwest University in China. He then worked as a university lecturer in Dalian University of Technology (2002-2005). In 2005 he joined Professor Alexandre G. Brolo’s group in University of Victoria for his PhD degree (2010). He moved back to China in 2011 and now is a full professor at the Department of Environmental Science and Engineering of the Southwest Jiaotong University. His research interests are mainly centered on surface-enhanced Raman scattering (SERS) and sustainable chemistry. "

Declaration of interests ☒ 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. ☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: