**4. SERS systems involving molecule aggregates**

Besides normal Raman, abundant SERS investigations have been undertaken by employing thin films of analytes on functional substrates, such as the extensive investigations of Raman and SERS from Langmuir-Blodgett (LB) films which are often associated with intermolecular interactions of aggregates [82–89]. One of the advantages is that the uniform sampling of thin films allows for better signal-to-noise of the SERS spectra from the analytes [82, 86]. Besides extensive investigations of such 2D assemblies of "analytes + nanoparticles" into thin films, previous publications also addressed 1D assemblies of molecular aggregates (analytes) and metal nanoparticles (signal amplifier).

#### **4.1. Template-based uniform assembly**

terpenoids, pinenes and their mixtures were examined showing distinguishable vibrational spectroscopic fingerprints of the three components respectively. It was noted that, in a certain case such as β-pinene, a dimer model reproduces the experimental results other than single molecule modeling, indicating nonneglectable intermolecular interactions and aggregation states for aerosols challenging the present mechanisms based on single molecule theory. Further, Raman spectra from an ambient sample can be analyzed using a hierarchical clustering method to check out whether the spectra of aerosols in consistence with relating organic compounds. In particular, analysis on time-resolved aerosol Raman spectra over the course of several hours, simply by checking the D-G bands of amorphous carbon plotted vs time (e.g., a half-hour intervals), enables to monitor and judge the increase/decrease of related pollution

**Figure 3.** (Top) Standard optical tweezers (Biral AOT 100) arrangement. (Inset) Valve system used to initiate exchange between D2O and H2O. (Bottom) A sketch showing isotopic water diffusion in aerosol by the use of optical tweezers.

Recently, Davies and Wilson [71] employed an aerosol optical tweezer technique for contactless levitation of single droplets (e.g., 3–6 μm in radius) and then for Raman investigations, as shown in **Figure 3**. Flexible environmental control system allows for rapid exchange of the gas-phase humidity source between H2O and D2O (**Figure 3**) to monitor the progression of the droplet composition using Raman spectroscopy. Utilizing a model describing diffusion in a sphere (i.e., solution to Fick's second law), they analyzed the data by varying diffusion coefficients (*D*w) in viscous media to achieve the best fit to both D2O and H2O data sets. This droplet-based isotopic tracer method takes a few advantages for measurement of diffusion coefficients. The resolution of gel formation suggests promising application to identify phase

in atmosphere [78, 80, 81].

38 Raman Spectroscopy and Applications

Reproduced with permission from Ref. [71].

Considering that Raman/SERS measurements at different positions of the samples could take on diversity due to molecule orientation and disorder degree, location and/or "hot-spots" dependence, the uniformity of molecular aggregates or SERS substrate is largely desired in order to get a better averaged collection of Raman signal. Anodic aluminum oxide (AAO) membrane is widely utilized as a versatile template to prepare 1D rodding/tubing and 2D nanoarray ordered structures, [90–95] both of which have found applications to SERS investigations. On one hand, AAO templates are ideal sublayers to filtrate and support noble metal nanoparticles hence forming highly SERS-active systems [96–98]. For another, AAO templates were also utilized to assemble organic molecules (e.g., perylene) for SERS investigations [11], as shown in **Figure 4**(left), where highly-ordered arrays of core-shell nano-pillars of Agperylene were fabricated simply by preparing perylene nanotubes utilizing the versatile AAO template [13], and followed by an electrochemical deposition of Ag [11]. Well-resolved Raman spectra with very good signal-to-noise background were obtained for the perylene (originally a large fluorescence yield) at an UV-vis excitation, profiting from the uniform assembly of perylene molecules. Based on the aforementioned theory and Eqs. (1) and (2), it was estimated that the molecular tilt angle is less than 54.7° indicating a head-to-tail J-aggregation of the perylene molecules along the inner walls of the AAO pores [11]. Similarly, high-quality SERS spectra of fullerene C60/C70 were also obtained from ordered arrays of core-shell nano-pillars of Au@C60/C70, as shown in **Figure 4**(right) [99]. These results evidenced that coincident and uniform assembly of fullerene molecules along the Au nano-rods leads to fluorescence quenching, and the ordered arrays of nano-pillars generate enhanced LSPR and hence remarkable SERS effect up to 10 times of signal amplification compared to the usual SERS results.

**Figure 4.** (Left) SERS spectrum of perylene from the standing Ag-perylene core-shell nano-pillars array; (middle) a sketch of the assembly of perylene molecules loaded with Ag as the core; (right) a sketch map showing the Au@C60/C70 nanopillars.

#### **4.2. Assemblies of "analytes + nanoparticles"**

By utilizing porous polymer monoliths functionalized with Ag nanoparticles as media, Liu et al. [100] showed a SERS system with assembly of the analyte molecules, as shown in **Figure 5**. The polymer monoliths composed of porous 3D-structured organic materials were prepared from monomers with unsaturated vinyl groups [101]. The monoliths could have μm- to nm- sized tortuous fluidic channel networks which enable the convection flow for rapid mass transfer while shorten the characteristic diffusion length. Compared to usual colloidal SERS systems, the monolith was demonstrated to concentrate the embedded metal nanoparticles and present a tremendous amount of surface area and more interaction between the analyte and Ag nanoparticles [100].

**Figure 5.** A sketch for the nanoparticle-functionalized porous polymer monolith detection elements for SERS investigations. Reproduced with permission from *Anal. Chem.* 2011 [100].

The one-dimensional assembly of "analytes + nanoparticles" may not need any supports or templates. For example, Z. Luo found that, micro-fiber assembly of organic molecules such as 2,2′-bipyridyl (22BPY) can be formed by directly injecting saturated solution of the target molecules into ice-cold Ag colloid [22]. This strategy was demonstrated to resemble a reprecipitation method (or named as microfluidic technique) which was widely used to prepare size-controlled organic nanostructures [102]. As shown in **Figure 6**, the high-quality spectrum suggested that the microfiber assemblies of 22BPY combined with Ag nanoparticles are a highly SERS-active systems differing from SERS of individuals [22].

**Figure 6.** (a) A SEM image of 22BPY microfibers via reprecipitation method by injecting saturated 22BPY solution into ice-cold Ag colloid, (b) microscopy bright-field image of a single fiber, and (c) correlative micro-Raman spectrum measured from this microfiber of 22BPY.

#### **4.3. On-the-specimen method**

**Figure 4.** (Left) SERS spectrum of perylene from the standing Ag-perylene core-shell nano-pillars array; (middle) a sketch of the assembly of perylene molecules loaded with Ag as the core; (right) a sketch map showing the Au@C60/C70

By utilizing porous polymer monoliths functionalized with Ag nanoparticles as media, Liu et al. [100] showed a SERS system with assembly of the analyte molecules, as shown in **Figure 5**. The polymer monoliths composed of porous 3D-structured organic materials were prepared from monomers with unsaturated vinyl groups [101]. The monoliths could have μm- to nm- sized tortuous fluidic channel networks which enable the convection flow for rapid mass transfer while shorten the characteristic diffusion length. Compared to usual colloidal SERS systems, the monolith was demonstrated to concentrate the embedded metal nanoparticles and present a tremendous amount of surface area and more interaction

**Figure 5.** A sketch for the nanoparticle-functionalized porous polymer monolith detection elements for SERS investiga-

The one-dimensional assembly of "analytes + nanoparticles" may not need any supports or templates. For example, Z. Luo found that, micro-fiber assembly of organic molecules such as 2,2′-bipyridyl (22BPY) can be formed by directly injecting saturated solution of the target molecules into ice-cold Ag colloid [22]. This strategy was demonstrated to resemble a reprecipitation method (or named as microfluidic technique) which was widely used to prepare size-controlled organic nanostructures [102]. As shown in **Figure 6**, the high-quality spectrum

nanopillars.

40 Raman Spectroscopy and Applications

**4.2. Assemblies of "analytes + nanoparticles"**

between the analyte and Ag nanoparticles [100].

tions. Reproduced with permission from *Anal. Chem.* 2011 [100].

As a powerful technique for trace analysis and detection due to the extremely high sensitivity and rich structural information that it can offer, SERS has been extensively investigated not only on the primal three classes of SERS systems (i.e., metal colloids, electrodes, and island films), but also non-traditional substrates [103–105]. For example, SERS studies involving surface coatings of Ag/Au nanoparticles, as named *on*-*the*-*specimen* method, have been largely applied in identification for art conservation, especially cultural relics and archeology [38, 106]. In general, the colorant components comprised of inorganic salts can be identified using normal Raman spectroscopic measurements, but strong fluorescence of organic dyes often precludes Raman measurements. SERS fulfills the requirements of an ideal analytical technique to detect and identify colorants and organic dyes in artworks [107–109]. In a few typical investigations such as those by Brosseau et al. [110, 111] examination on the samples of actual historical textiles, pastels, and watercolors, etc. have been conducted revealing the unique advantages of SERS sensitivity and providing distinguishable information available for long term preservation. Recently utilizing bubbling gas strategy for laser ablation in liquid (LAL), Luo et al. [112] prepared chemically-pure gold clusters for a practical use of discrimination among different surfaces, as demonstrated the identification of various documents from different printers/copiers and written with different pen-inks, as shown in **Figure 7** [112]. These investigations pertaining to molecule aggregation states give important application of Raman/ SERS spectroscopy within a minimally-invasive manner [112].

**Figure 7.** (A) Undistinguishable Raman of a "PSU" document from both a printer and a copier (a); SERS spectrum of the document from the printer (Xerox Phaser 8560DN PS, Genuine XEROX Solid Ink, black) (b), compared with that from a copier (RICOH, Aficio, MP 7001) (c). (B) SERS examination of four handwritten samples, by coating the gold clusters on the ink-area.

Recently, Tian et al. have further pullulated this method in help of shell-isolated nanoparticleenhanced Raman scattering (SHINERS) technique [113, 114], as sketched in **Figure 8**. For a typical SHINERS system, Au nanoparticles were coated with ultrathin silica shells and sowing on probed surfaces, where the Au core provides SERS signal enhancement while the silica shell shields the metal core from direct contact with analyte molecules (i.e., prevents the contamination of the chemical system under study) [115], which differs from general SERS sampling method, simply by adding analytes onto SERS-active substrates or directly mixing the target solution with metal colloids [115–118]. The SHIERS 'smart dust' on the analyte surfaces was demonstrated of practical use in a few interdisciplinary research fields, such as inspecting pesticide residues on food and fruit, examining drug security and environment protection accurately and rapidly, and characterizing biological structures.

**Figure 8.** A sketch showing the in-situ probing of biological structures by SHINERS. Reproduced with permission from *Nature* 2010. [61, 62]
