**3. SERS active substrates**

based on the charge transfer effect of the adsorbed molecule on the enhancement of Raman signal [12]. This is known as the chemical enhancement. This chemical enhancement theory depends on the charge transfer complex formation of the adsorbed molecule by absorption of photon of the suitable wavelength. Nevertheless, it is extremely complicated to separate these

In the mid-1980s, the spotlight on SERS research diverted to the exploitation of SERS effect for new and novel analytical and biological applications from the basic understanding of the mechanism responsible for the SERS phenomenon. However, it was extremely difficult to record high-quality, highly reproducible and stable SERS spectra as demonstrated by some of the investigations carried out in the mid-1980s as well as the early 1990s. The SERS spectra obtained were highly irreproducible, which can be explained by the small variations in the fabrication of SERS substrates. This shortcoming has prevented the development of SERS as a quantitative tool for long period. For that reason, fabrication of an SERS active substrate is very important in SERS research so that highly uniform, stable, and highly reproducible SERS

In the mid-1990s, VIIIB transition metals were employed as SERS active substrates to carry out electrochemical surface-enhanced Raman spectroscopy (EC-SERS) and these SERS substrates were further utilized for electrochemistry and catalysis [13]. Professor Tian and his coworkers at the Xiamen University, China, first introduced quite a few surface roughening procedures and demonstrated that the SERS effect can be directly obtained from transition metals such

signals can be obtained.

298 Raman Spectroscopy and Applications

**Figure 4.** The photograph of Fleischmann, the inventor of SERS.

two effects experimentally and understand the overall mechanism of SERS.

The metals that are selected to be used as an SERS active substrate can be determined by the plasmon resonance frequency. Both, visible and near-infrared radiation (NIR) can generally be used for excitation of the Raman modes. Silver (Ag), gold (Au), and copper (Cu) are typically utilized for carrying out SERS experiments because their plasmon resonance frequencies are in the region of above-mentioned wavelength of electromagnetic spectrum, providing maximal enhancement for visible and NIR light [5–7]. Recently, transition metals such as platinum (Pt), ruthenium (Ru), palladium (Pd), iron (Fe), cobalt (Co), and nickel (Ni) have been utilized as SERS active substrates. These transition metals can display enhancements between 1–3 orders of magnitude and these enhancement factor values are very low compared to the enhancement factors obtained for metals such as Au and Ag. This can be explained by the fact that excitation of the surface plasmon resonances in the visible light region is extremely difficult. Nevertheless, large enhancement (~ 10<sup>4</sup> ) values can be obtained from the transition metals using excitation wavelength in the near ultra-violet (UV) region. The major features of the SERS technique are abridged briefly as follows:

• SERS is highly surface sensitive, nondestructive and *in-situ* vibrational spectroscopic analytical technique.


Therefore, the fabrication of an SERS active substrate is a very important field from the point of view of SERS research. The two most commonly used SERS substrates are metallic colloids of Au, Ag, and Cu obtained from chemical reduction and the metal electrode surfaces roughened by one or more electrochemical oxidation-reduction cycles. Surface and substrate generality are the major limitations associated with the SERS effect and many research groups all over the world have tried to surmount these two major problems by obtaining SERS activity from other metallic surfaces other than Ag, Au, and Cu and from atomically flat (single crystal) surfaces rather than roughened surfaces. However, most of the metals used as SERS substrates will exhibit poor biological compatibility. For that reason, it is essential to provide new novel substrates for SERS study. For an ideal SERS substrate, the material should be economical, easily accessible, chemically inert, as well as biocompatible.

The SERS substrates can be approximately divided into three categories: (1) metal nanoparticles (MNPs) in suspension, (2) metal nanoparticles immobilized on solid substrates, and (3) metal nanostructures fabricated directly on solid substrates by nanolithography and template based synthesis [7, 17]. Although both nanoparticles and nanoparticle film electrodes can exhibit good surface uniformity, as a result difficulty in controlling the spacing of the nanoparticles will not be able to optimize the SERS activity. Only template-based fabrication methods can be employed to obtain highly ordered SERS substrates with controlled interparticle spacing. Amid different template-based methods, nanosphere lithography (NSL), and anodic aluminum oxide (AAO) films are most commonly employed to fabricate highly ordered SERS active substrates. The Langmuir-Blodgett technique can also be utilized to fabricate highly ordered SERS active substrates. **Figure 5** illustrates the different SERS active substrates used in recent years. As a result of fast advancement in nanoscience and nanotechnology, several methods are accessible now for the fabrication of various metallic nanostructures with different size, shape, which can be further utilized as SERS active substrates. This has facilitated in a significant enhancement in the citations of new SERS active substrates available in the literature in the last 5 years and we are expecting a further improvement in the upcoming years.

Metal nanoparticles in suspension are the simplest of all SERS active substrates used so far, where the SERS effect can be studied in the presence of definite concentration of analytes (Raman probe molecules). However, aggregation of metallic nanoparticles can prevent to obtain highly reproducible SERS spectra. Alternatively, aggregation is sometimes essential for obtaining high quality, highly reproducible SERS signal [15]. MNPs suspension must be mixed with the analyte solution for carrying out the SERS experiment, a sampling requirement that might be limiting for a few real-life applications, such as quantitative analysis of adsorbates on nonSERS active surfaces like semiconductors and fruits. In spite of the problems such as reproducibility in experimental results and potential sampling, MNPs suspensions are extensively employed as an SERS active substrate due to their high SERS-performance, good stability, and simplicity in production. Actually, this kind of substrate was employed in the early years for carrying out single molecule SERS experiments. The drawback of sampling has been recently overcome by Professor Tian and his research group at the Xiamen University, China [18]. They introduced a completely original shell-isolated MNPs as an SERS enhancing smart dust, which was successfully employed for probing hydrogen adsorbed on the single crystal Pt surface, and direct detection of pesticides residues in the form of contamination in citrus fruits such as oranges. This new borrowing SERS technique is referred by them as shellisolated nanoparticle-enhanced Raman spectroscopy (SHINERS) [18].

• SERS occurs as a result of bringing the Raman probe molecules closer within the few nano-

• The SERS technique will exhibit exceptionally high spatial resolution, in which the enhancement range is several nanometers, effective for one or several molecular layers

• SERS activity is strongly dependent on the type of metal and surface roughness of the SERS

Therefore, the fabrication of an SERS active substrate is a very important field from the point of view of SERS research. The two most commonly used SERS substrates are metallic colloids of Au, Ag, and Cu obtained from chemical reduction and the metal electrode surfaces roughened by one or more electrochemical oxidation-reduction cycles. Surface and substrate generality are the major limitations associated with the SERS effect and many research groups all over the world have tried to surmount these two major problems by obtaining SERS activity from other metallic surfaces other than Ag, Au, and Cu and from atomically flat (single crystal) surfaces rather than roughened surfaces. However, most of the metals used as SERS substrates will exhibit poor biological compatibility. For that reason, it is essential to provide new novel substrates for SERS study. For an ideal SERS substrate, the material should be eco-

The SERS substrates can be approximately divided into three categories: (1) metal nanoparticles (MNPs) in suspension, (2) metal nanoparticles immobilized on solid substrates, and (3) metal nanostructures fabricated directly on solid substrates by nanolithography and template based synthesis [7, 17]. Although both nanoparticles and nanoparticle film electrodes can exhibit good surface uniformity, as a result difficulty in controlling the spacing of the nanoparticles will not be able to optimize the SERS activity. Only template-based fabrication methods can be employed to obtain highly ordered SERS substrates with controlled interparticle spacing. Amid different template-based methods, nanosphere lithography (NSL), and anodic aluminum oxide (AAO) films are most commonly employed to fabricate highly ordered SERS active substrates. The Langmuir-Blodgett technique can also be utilized to fabricate highly ordered SERS active substrates. **Figure 5** illustrates the different SERS active substrates used in recent years. As a result of fast advancement in nanoscience and nanotechnology, several methods are accessible now for the fabrication of various metallic nanostructures with different size, shape, which can be further utilized as SERS active substrates. This has facilitated in a significant enhancement in the citations of new SERS active substrates available in the literature in the last 5 years and we are expecting a further improvement in

Metal nanoparticles in suspension are the simplest of all SERS active substrates used so far, where the SERS effect can be studied in the presence of definite concentration of analytes (Raman probe molecules). However, aggregation of metallic nanoparticles can prevent to obtain highly reproducible SERS spectra. Alternatively, aggregation is sometimes essential for obtaining high quality, highly reproducible SERS signal [15]. MNPs suspension must be mixed with the analyte solution for carrying out the SERS experiment, a sampling require-

meters of the surface of SERS substrates of different morphologies.

nomical, easily accessible, chemically inert, as well as biocompatible.

close to the SERS active substrate.

300 Raman Spectroscopy and Applications

the upcoming years.

active substrate employed for the study.

**Figure 5.** The different SERS active substrates fabricated by nanotechnology. Reproduced with permission from Haynes et al. [36]. Copyright @ American Chemical Society. (a) Rough silver film on glass; (b) Silver coating on top of PS beads; (c) Nanosphere lithography; (d) Silver column produced by e-beam lithography. after the word by nanotechnology

For crucial evaluation, the SERS substrates covered in this review article can be limited to only three general types of substrates, classified according to their fabrication technique: (1) MNPs immobilized in planar solid supports, (2) metallic nanostructures fabricated using nanolithographic methods, and (3) metallic nanostructures fabricated using template-based techniques.

The dispersed and aggregated MNPs cannot be used as a SERS active substrate in real analytical problems as a result of the poor reproducibility of SERS enhancement factor, which can be solved by immobilizing the MNPs on some kind of solid support. Since the first report of an SERS substrate consisting of MNPs synthesized by a wet chemical method and afterward immobilizing them onto a solid support [19], the process has become extremely popular and several papers have been published based on this process as found from the literature survey.

The top-down nanolithography and associated nanoimprint lithographic-based fabrication techniques were employed to fabricate highly ordered metallic nanostructures array. In this technique, a layer of polymeric photoresist (positive or negative) is cast on the solid substrates (such as Si, glass, or Au film). It was followed by direct patterning on the photoresist surface or indirect patterning with the assistance of a mold using ultraviolet (UV) light, an electron beam, or a focused ion beam. Afterward, the residual photoresist can be utilized as a mold, on which SERS-active metals are deposited by a physical vapor deposition technique under vacuum conditions. The mold was lifted off and a highly ordered nanostructured SERS-active substrate with a structure identical or complementary to that of the mold is formed. Highly ordered and uniform SERS active substrates with interparticle spacings below 10 nm can be produced by employing the nanolithographic method with a broad variety of shapes and geometries. However, nanolithographic techniques are still time-consuming and very expensive due to the use of high energy focused ion beam (FIB) or electron beam (EB) for the fabrication of the SERS substrate with a large area. Both, FIB and EB lithographic techniques can be employed to make molds for the nanoimprint lithography technique. In the nanoimprint lithographic technique, the desired nanopatterns are produced by direct writing on the Si or quartz slide by using an electron beam, which can be subsequently used as the mold. Next, the mold is aligned and pressed into the photoresist covering on the substrate and finally, the mold is lifted off after curing. Subsequently, the substrate is deposited with the desired metal to be used as an SERS active substrate. A highly ordered nanostructure with good SERS activity can be fabricated by completely removing the photoresist. Nanoimprint lithographic techniques are more efficient and inexpensive as compared to nanolithographic techniques.

Van Duyne and his group developed the nanosphere lithographic technique in 2001 [20, 21]. In this technique, highly ordered single or multi-layer colloidal crystal templates are produced from the self-assembly of monodispersed polystyrene or SiO2 nanospheres of the desired diameter on clean conducting substrates such as indium tin oxide (ITO) or evaporated metal substrate over glass. Afterward, a metal layer is deposited by the physical vapor deposition or electrochemical deposition method on the substrate with a controlled thickness. Three types of structured SERS substrate can be produced by the nanosphere lithographic technique: (1) Ag metal "film over nanosphere" (FON) surfaces are formed due to physical vapor deposition on the nanosphere template; (2) surface confined nanoparticles with a triangular footprint are produced by the removal of nanospheres of the FON surfaces by sonication in a solvent; (3) thin nanostructured films containing regular hexagonal array of uniform segment sphere voids are formed by electrochemical deposition followed by removal of spheres. One can control the shape, size, and interparticle spacing of the fabricated nanostructures by tuning the size of the nanospheres and the thickness of the deposited metal with the ultimate objective that the localized surface plasmon resonance (LSPR) position can be adjusted to match the excitation wavelength with an optimized SERS enhancement.

Arrays of silver (Ag) nanoparticles with a precisely controlled gap up to 5 nm are electrochemically grown by Wang et al. [22] by utilizing the porous AAO film as a template material. This Ag/AAO system with tunable sub-10 nm interparticle gap can be further utilized as highly ordered, uniform SERS active substrate with high value of SERS enhancement factor (~10<sup>8</sup> ). It is extremely difficult to precisely control of interparticle gaps between nanostructures on an SERS-active substrate in the sub-10 nm regime as known from the existing literature. Such studies are crucial for the fabrication of SERS active substrates with uniformly high enhancement factors, and for overall understanding of collective surface plasmons existing inside the gaps. The "hot junction" or "hot spot" located at the interparticle gap of these nanostructure-based SERS substrates can enhance the SERS activity, which is an important aspect for large electromagnetic field enhancement and excellent SERS sensitivity. Wang et al. experimentally demonstrated the first quantitative study of the collective SERS effect on a substrate with precisely controlled "hot junctions" in the sub-10 nm regime, and confirmed the theoretical prediction of interparticle-coupling-induced Raman enhancement. This Ag/AAO-based SERS substrates fabricated by Wang et al. [22] with highly uniform and reproducible SERS signals can be utilized as both a biosensor and a chemical sensor with extremely high sensitivity. Using these SERS substrates, concentration up to picomolar level has been detected. This excellent SERS substrate can be further exploited for single molecule SERS study increasing the overall detection limit and SERS sensitivity.

The top-down nanolithography and associated nanoimprint lithographic-based fabrication techniques were employed to fabricate highly ordered metallic nanostructures array. In this technique, a layer of polymeric photoresist (positive or negative) is cast on the solid substrates (such as Si, glass, or Au film). It was followed by direct patterning on the photoresist surface or indirect patterning with the assistance of a mold using ultraviolet (UV) light, an electron beam, or a focused ion beam. Afterward, the residual photoresist can be utilized as a mold, on which SERS-active metals are deposited by a physical vapor deposition technique under vacuum conditions. The mold was lifted off and a highly ordered nanostructured SERS-active substrate with a structure identical or complementary to that of the mold is formed. Highly ordered and uniform SERS active substrates with interparticle spacings below 10 nm can be produced by employing the nanolithographic method with a broad variety of shapes and geometries. However, nanolithographic techniques are still time-consuming and very expensive due to the use of high energy focused ion beam (FIB) or electron beam (EB) for the fabrication of the SERS substrate with a large area. Both, FIB and EB lithographic techniques can be employed to make molds for the nanoimprint lithography technique. In the nanoimprint lithographic technique, the desired nanopatterns are produced by direct writing on the Si or quartz slide by using an electron beam, which can be subsequently used as the mold. Next, the mold is aligned and pressed into the photoresist covering on the substrate and finally, the mold is lifted off after curing. Subsequently, the substrate is deposited with the desired metal to be used as an SERS active substrate. A highly ordered nanostructure with good SERS activity can be fabricated by completely removing the photoresist. Nanoimprint lithographic techniques are more efficient and inexpensive as compared to nanolithographic techniques.

Van Duyne and his group developed the nanosphere lithographic technique in 2001 [20, 21]. In this technique, highly ordered single or multi-layer colloidal crystal templates are produced

diameter on clean conducting substrates such as indium tin oxide (ITO) or evaporated metal substrate over glass. Afterward, a metal layer is deposited by the physical vapor deposition or electrochemical deposition method on the substrate with a controlled thickness. Three types of structured SERS substrate can be produced by the nanosphere lithographic technique: (1) Ag metal "film over nanosphere" (FON) surfaces are formed due to physical vapor deposition on the nanosphere template; (2) surface confined nanoparticles with a triangular footprint are produced by the removal of nanospheres of the FON surfaces by sonication in a solvent; (3) thin nanostructured films containing regular hexagonal array of uniform segment sphere voids are formed by electrochemical deposition followed by removal of spheres. One can control the shape, size, and interparticle spacing of the fabricated nanostructures by tuning the size of the nanospheres and the thickness of the deposited metal with the ultimate objective that the localized surface plasmon resonance (LSPR) position can be adjusted to match the

Arrays of silver (Ag) nanoparticles with a precisely controlled gap up to 5 nm are electrochemically grown by Wang et al. [22] by utilizing the porous AAO film as a template material. This Ag/AAO system with tunable sub-10 nm interparticle gap can be further utilized as highly ordered, uniform SERS active substrate with high value of SERS enhance-

nanostructures on an SERS-active substrate in the sub-10 nm regime as known from the

). It is extremely difficult to precisely control of interparticle gaps between

nanospheres of the desired

from the self-assembly of monodispersed polystyrene or SiO2

excitation wavelength with an optimized SERS enhancement.

ment factor (~10<sup>8</sup>

302 Raman Spectroscopy and Applications

The Langmuir-Blodgett (LB) technique can be employed for the large-scale production of a fully defect-free SERS substrate over a large area of few hundred of cm2 . The LB method was initially employed to form a large-area surface film of amphiphilic molecules on solid substrates. In this procedure, the amphiphilic molecules such as stearic acid is dissolved in a volatile solvent like benzene or chloroform, which is completely immiscible with water, and soon after, the solution is dispersed on the surface of the water phase. As a result, a monomolecular film of the amphiphilic molecule will form at the air/water interface with complete evaporation of the volatile solvent. The film can be deposited on the substrate by the dipping and pulling method. Similarly, a nanoparticle film can be fabricated by the LB method. In this procedure, the surfaces of nanoparticles are modified with hydrophobic molecules and dispersed directly into a highly volatile solvent, which is immiscible with water. By dispersion of the solution into the water phase, a layer of randomly distributed nanoparticles will be left at the interface after complete evaporation of the volatile solvent. By compressing the layer through moving the barrier, one can control the density of the monolayer film. Subsequently, an ordered layer of nanoparticles will be formed on the surface. A systematic SERS study was carried out by Yang and his group using an SERS active substrate fabricated by the LB technique [23]. The LB technique was employed by them to successfully fabricate most uniform SERS substrates of films of nanorods, nanowires, and spherical, cubic, cuboctahedral and octahedral Ag nanoparticles [23].
