Titanium (IV) Oxide Nanotubes in Design of Active SERS Substrates for High Sensitivity Analytical Applications: Effect of Geometrical Factors in Nanotubes and in Ag-n Deposits Titanium (IV) Oxide Nanotubes in Design of Active SERS Substrates for High Sensitivity Analytical Applications: Effect of Geometrical Factors in Nanotubes and in Ag-n Deposits

In this chapter, we summarize the results of recent investigations into TiO 2 nanotubu- lar oxide layers on Ti metal loaded with Ag nanoparticles, which act as efficient sur face plasmon resonators. These Ag-n/TiO 2 NT/Ti composite layers appear to be useful as platforms for precise surface analytical investigations of minute amounts of numerous types of organic molecules: pyridine (Py), mercaptobenzoic acid (MBA), 5-(4-dimethyl- aminobenzylidene) rhodamine (DBRh) and rhodamine (R6G); such investigations are known as surface enhanced Raman Spectroscopy (SERS). Geometrical factors related to the nanotubes and the silver deposit affect the SERS activity of the resulting com posite layers. The results presented here show that, for a carefully controlled amount of Ag-n deposit located mainly on the tops of titania nanotubes, it is possible to obtain high-quality, reproducible SERS spectra for probe molecules at an enhancement factor of 10 5 –10 6 . This achievement makes it possible to detect organic molecules at concentrations as low as, e.g., 10 −9 M for R6G molecules. SEM investigations suggest that the size of the nanotubes, and both the lateral and perpendicular distribution of Ag-n (on the tube tops and walls), are responsible for the SERS activity. These features of the Ag-n/TiO 2 NT/Ti composite layer provide a variety of cavities and slits which function as suitable resona- tors for the adsorbed molecules.


Introduction
Searching for new and effective substrates for surface enhanced Raman Spectroscopy (SERS) applications is a subject of research at many scientific centers. This is because SERS spectroscopy is used to quickly characterize many functional materials important in advanced technologies, analytical chemistry and biology [1]. An important practical problem when carrying out analytical SERS measurements is to obtain suitable substrates containing a sufficient number of electromagnetic nanoresonators. The SERS effect is strongly dependent on an enhancement of the Raman scattering intensity by molecules adsorbed on a nanostructured metallic surface. The SERS enhancement factor is related to the size and shape of the nanostructures, which causes electromagnetic enhancement [2,3]. Usually, the average value of SERS enhancement is around 10 5 -10 6 , but localized enhancement may reach values of 10 10 at certain, highly efficient sub-wavelength regions of the surface [4]. It is therefore desirable for the resulting substrates to render a very large SERS spectra enhancement factor, where the enhancement factor thus obtained does not depend on where the measurement is made (the same enhancement factor should be present at different locations on the sample surface [5]).
A promising substrate for SERS measurements that meets these two most important practical requirements is regular TiO 2 nanotubes loaded with metal nanoparticles, which support surface plasmon resonance. One way of modifying the nanotubular layer and attaining high SERS activity is to enhance the surface with a small quantity of Ag or other plasmonic metal (e.g. Au or Cu) nanoparticles (plasmonic nanoparticles) [1,6]. Such plasmonic metals themselves are known for their high SERS activity, which results only after a proper roughening of their surfaces [7]. The dielectric constant for these metals consists of a negative real part and a small positive imaginary part. When a nanoparticle made of these metals interacts with electromagnetic radiation, collective oscillations of the surface plasmons are induced [8]. These oscillations result in an enhanced electromagnetic field in close proximity to the surface of the nanoparticles. The surface of these metals, when properly roughened, provides suitable slits and cavities which serve as surface plasmon resonators that strongly enhance the intensity of the electromagnetic field. For Raman bands with a small Raman shift, the increase in the efficiency of Raman scattering is roughly proportional to the fourth power of the field enhancement [2,9]. A detailed description of the SERS effect can be found in literature [2,[9][10][11][12].
It is well known that the enhancement factor (E F ) of SERS spectra depends on the metal morphology/topography. Only a suitable surface roughness of the SERS substrates can produce a stronger Raman signal. The optimum value of the size and shape of the noble metal particles may lead to a maximum E F (which also depends on the nature of the metal, the excitation laser wavelength, and special experimental conditions) [13]. A highly active substrate provides superior conditions for measuring the SERS spectra of an adsorbate. Therefore, to gain some insight into the substrate-adsorbate interactions at the molecular level, and to detect different kinds of organic adsorbates, detailed knowledge of how to fabricate a highly sensitive and reproducible SERS substrate is of considerable importance [1,5].
The high regularity of the TiO 2 nanotubular structures obtained ensures that the enhancement factors are high reproducible [14][15][16]. The specific morphology of the resulting structures (the large side wall surfaces of the nanotubes grown perpendicular to the substrate) makes it possible to prepare metallic nanograin systems with a large number of gaps between the nanotubes (i.e. the metal nanoparticle systems are close to each other on the side walls of TiO 2 nanotubes that are perpendicular to the macroscopic Ti surface). As shown experimentally, the narrow gaps between the metal nanoparticles supporting surface plasmon resonance produce the largest SERS signal enhancement factors [17][18][19] (see Figure 1).
In this chapter we discuss in more detail the effect of a TiO 2 nanoporous structure with Ag nanoparticles deposited by different PVD methods (which provide precise control over the amount of silver sputtered onto the nanotubular substrate) on the SERS activity of the substrates prepared. We focus on the geometrical effects on SERS activity: nanotube diameter, size and distribution of Ag-n, and morphology of the Ag-n agglomerates. TiO 2 nanotubes are particularly suitable for such investigations because the strictly controlled electrochemical procedures make it possible to produce well-formed, nanotubular substrates that are "homogeneous," statistically having the same nanotube diameter and nanotube wall thickness (see Figure 2).  As shown in Figure 2, the specific morphology of TiO 2 nanotubes can be relatively easily modified by controlling the conditions of anodic polarization (type of electrolyte, voltage and anodization time), because there is a direct linear relationship between anodization voltage and the average diameter of the nanotubes formed. In general, the diameter and wall thickness of the nanotubes increase with anodic voltage [20,21]. The possibility of preparing nanotubes of different size, shape and wall thickness provides control over the geometrical surface area and specific surface area, which are important parameters when developing new substrates for SERS applications.

Fabrication, surface and structure characterization of SERS substrates based on TiO 2 nanotubes
Innovative SERS active platforms based on TiO 2 NT with noble metal deposits (Ag) were used for investigating various organic probe molecules such as pyridine (Py), mercaptobenzoic acid (MBA), organic dye 5-(4-dimethylaminobenzylidene) rhodamine (DBRh) and rhodamine 6G (R6G). For this purpose, simple electrochemical methods were applied: anodic oxidation of Ti foil (0.25 mm-thick, 99.5% purity, Alfa Aesar) in an optimized electrolyte: a glycerol/ water mixture (volume ratio 50:50) with 0.27 M NH 4 F under different constant voltages from 10 up to 30 V. They led to the formation of nanoporous titanium oxide structures, and subsequently, to the preparation of specific metal nanostructures on surfaces thereof during PVD processes (magnetron sputtering, evaporation at high and low vacuum). Before the Ag deposition, all of the samples were annealed in air at 650°C for 3 h in order to transform the structure of the TiO 2 NT from amorphous to crystalline [19,22].
Ag nanoparticles (from 0.01 up to 0.03 mg/cm 2 ) were deposited using the sputter deposition technique: the evaporation method in a low vacuum (p = 3 × 10 −3 Pa) with a JEE-4X JEOL device, and the DC magnetron sputtering technique using a Leica EM MED020 apparatus in a configuration perpendicular to the surface of the samples. More details are given elsewhere [18,23]. We strictly controlled in situ the average amount of metal deposited per cm 2 using a quartz microbalance. One has to consider, however, that the true local amount of the metal deposits may vary substantially from site to site. The highly developed specific surface area of the nanotube arrays and its brush-like morphology may strongly affect Ag local distribution, resulting in considerable non-uniformity. For this type of process, silver targets of 99.9% purity (Kurt J. Lesker Company) were used. To better control the silver sputtering deposition process on the surface of the nanotubes, we applied the thermal evaporation method (0.01 and 0.05 mg/cm 2 ) using an EF 40C1 effusion cell inside an UHV preparation chamber (PREVAC, Poland). The cell was maintained at a temperature of 900°C during the process of resistive evaporation. Silver (2 mm-diameter wire, 99.999%, Alfa Aesar) was evaporated onto the room temperature-surface of the TiO 2 NT at a pressure of 1-2•10 −6 Pa at a constant evaporation rate of 0.13 nm/min. The evaporation rate from the silver effusion cell was calibrated and monitored using a TM-400 quartz crystal thickness monitor (Maxtek Inc.). The process of fabricating the SERS substrates is presented schematically in Figure 1.
The SERS platforms thus fabricated were characterized using various analytical methods. Electron Microscopy was applied for the morphological and structural characterization of the nanotubes after each preparation stage. The SEM observations (FEI NovaNanoSEM 450, Hitachi S70, Hitachi S-5500) were carried out at an accelerating voltage of 5 or 10 kV and an SE detector to reveal topographic contrast at the tops of the tubes. The thin sample for STEM observations was prepared by using the lift-out technique with a Hitachi NB-5000Focused Ion Beam system. The internal structure of the nanotubes was examined with a dedicated STEM Hitachi HD2700 in BF and atomic mass contrast. Observations at 200 kV revealed a columnar structure of the nanotubes and the transition zone. The surface chemical composition and the chemical state of the surface species (Ti, O, Ag) of the fabricated SERS platforms were examined using XPS spectroscopy. The XPS spectra were measured with a Microlab 350 Thermo Electron spectrometer at 300 W non-monochromatic Al K α radiation and 1486.6 eV energy. AES spectroscopy was applied to determine the local chemical composition of the SERS substrates obtained. All of the spectra were recorded at an energy of 10 kV. The appropriate standards for AES and XPS reference spectra were also used. Finally, the Raman spectra of 2-mercaptoethanesulfonate (5 × 10 −3 M), pyridine (5 × 10 −2 M, in a mixture of pyridine and 0.1 M KCl), DBRh (10 −4 M, in a mixture of water and ethanol (1:2)), and rhodamine from a 10 −7 and 10 −9 M aqueous solution were collected with a Horiba Jobin-Yvon Labram HR800 using a He-Ne laser (632.8 nm) as the excitation source. For each sample, 20 measurements were performed locally at various points chosen randomly.

Results and discussion
For our studies, we used an optimized electrolyte based on a mixture of glycerol and water (volume ratio 50:50) with 0.27 M NH 4 F. The growth of nanotubes under a constant voltage is perpendicular to the metal substrate, as shown in Figure 3. STEM images reveal nanotubes that are hollow in shape, separated from each other, and that feature a characteristic "columnar structure." A typical surface morphology of the nanotubes is shown in the inset to Figure 3 (10 and 25 V). Usually, the as-grown porous anodic layers exhibit poor adhesion to the Ti substrate, and so, to improve their adhesion and mechanical stability, the samples were annealed in air at 650°C for 3 h. The heat treatment in this temperature range does not cause any visible changes in the diameter or shape of the TiO 2 nanotubes, but in the annealing process an interfacial region is formed between the Ti substrate and the TiO 2 nanotubes that stabilizes the entire TiO 2 NT/Ti substrate system. Moreover, the heat treatment results in a change in the nanotube structure, from amorphous (directly after anodization) to crystalline: anatase [19,21,24]. All these factors are crucial for the suitability of the nanotubes as substrates when preparing SERS-active adsorbates.
After annealing, the above structures proved to be important for designing active substrates for SERS spectroscopy, where a large surface area and a stabilized structure are required [19]. The free-standing nanotubes adorned with Ag nanoparticles formed a layer of natural nanoresonators (antennas), which repeatedly enhanced Raman scattering [14,18]. However, the geometrical factors of the Ag-n deposit and their relation to the geometry of the nanotubes are not yet well understood. Therefore, the details of the SERS mechanism should be carefully considered.
In general, there are two important mechanisms underlying SERS. The first, and the dominant, mechanism toward large SERS enhancement factors (E F ) is that of electromagnetic field enhancement, where localized surface plasmons (LSPs) in the metallic nanostructure increase the Raman signal intensity. The other contribution to SERS E F is the chemical enhancement mechanism, where the charge transfer between the adsorbed molecule and the metal plays a critical role in enhancing and modifying the modes of molecular vibration [25]. Both enhancement mechanisms can operate simultaneously when using TiO 2 nanoporous structures in the form of freestanding nanotubes with a suitable, carefully prepared deposit of silver nanoparticles.  agglomerated and form rings. As a result of silver evaporation at a low vacuum, the plasmonic nanoparticles are distributed homogenously in the TiO 2 nanotube layer, tightly covering the walls of the nanotubes and forming a thin, solid coating around the tubes (Figure 4b). After evaporation at a high vacuum, where the process of silver deposition is very slow (0.13 nm/min), spherical Ag nanoparticles are formed (see Figure 4c). The diameter of these particles is below 50 nm. Densely-packed TiO 2 nanotubes coated with a plasmonic metal (Ag) could act as antennananoresonators having a strictly defined geometry and surface development. This would ensure the formation of highly active places-"hot spots," i.e., gaps and cavities serving as surface plasmon resonators that significantly increase the intensity of the electromagnetic field.   In order to gain further insight into the chemical state of the samples before and after the silver deposition processes, XPS measurements were performed for those samples having a small amount of Ag (0.01 mg/cm 2 ) (see Table 1). The deconvolution of the main XPS signals for Ti2p 3/2 and O1s suggests that titanium is bound to oxygen and forms titanium oxide IV. Highresolution XPS spectra confined to the Ag range gave the binding energies of Ag3d 5/2 peaks located at 367.9 (high vacuum), 368.3 (magnetron sputtering) and 368.4 eV (low vacuum), respectively, which is consistent with the literature [26][27][28][29][30] and also with our reference data. This implies that the Ag agglomerates and single nanoparticles located on the tops and in the deeper parts of the TiO 2 nanotubes are metallic silver. A small shift in the Ag3d 5/2 and Ti2p 3/2 peaks (see Table 1) for the samples after functionalization by PVD methods (±0.1-0.4 eV) may suggest that the XPS signals of Ag are modified by an interaction with the TiO 2 nanoporous substrate in relation to the position of the Ag standard peak. This interaction may induce a shift in the Fermi level in the deposited silver, in particular, if single nanoparticles are supported on the oxide carrier -the SMSI effect. Such effects have been reported by Goodman et al. and Lopez et al. [31,32] for gold nanoparticles on TiO 2 supports, and were also observed in our recent work, where a ZrO 2 nanoporous layer was covered with Ag nanoparticles [33]. Figure 6 shows the SERS spectra of pyridine (Py) adsorbed at TiO 2 NT/Ti platforms fabricated at 25 V and covered with the same average amount of Ag-n (0.01 mg/cm 2 ) by the three different procedures: magnetron sputtering, and vacuum evaporation at low and high vapor pressures. The spectra presented were averaged from 20 measurements on each sample tested. The SERS spectra of pyridine are dominated by two bands: at ~1010 and at ~1034 cm −1 originating from the aromatic ring vibrations of this molecule. Moreover, on the spectra recorded, there are also others bands clearly visible at 1150 and 1220 cm −1 , which are also characteristic of Py adsorbed on a standard silver surface [34,35]. The SERS measurements revealed that the distribution of Ag nanoparticles on the nanotubular substrate affects SERS intensity (compare

Figure 4). A roughly two-fold increase in SERS intensity occurs when the titania nanotubes
are adorned with Ag-n after the magnetron sputtering process. Three kinds of Ag particles can be distinguished: those accumulated on the tops of the nanotubes, forming "rings," and single particles (see. Figure 4) separated from each other and/or those Ag particles produced in the high vacuum process, which are located on the tops ("mouth") of the nanotubes. The latter do not yield such a strong SERS effect. Apparently, the key factor in the SERS activity of Ag-n is the size and mode of the specific surface area of the silver particles formed during a specific vacuum process. While the SERS intensity reached was not very high, well-reproducible and good-quality SERS spectra were obtained.
The next step was to repeat the same measurements with a nanotubular substrate prepared at an anodization of 10 V to produce nanotubes having a smaller diameter, thereby increasing the specific surface area of the SERS-active silver.   bands at 1004 and 1034 cm −1 . The band at 1004 cm −1 is due to the ring breathing mode (ν 1 , Wilson notation), whereas that at 1034 cm −1 is due to symmetric triangular ring deformation (ν 12 ) [34,35]. The SERS spectral intensity increases distinctly with the amount of Ag metal deposit, which correlates with a change in surface topography (compare Figure 7). This effect is most likely related to an increase in the number of narrow gaps between the silver particles themselves (locations particularly active in SERS spectroscopy) with an increasing amount of Ag deposit.
Our previous experiments show that the SERS enhancement factor for free-standing TiO 2 nanotubes adorned with silver nanoparticles is strictly related to the size of the nanotubes. The SERS enhancement factor (E F ) for the pyridine (Py) probe molecule was successfully estimated using the following formula (1): where I SERS and I ref are the Raman intensities obtained from the SERS and normal Raman (NR) investigations, respectively, c ref stands for the concentration of pure Py in the NR measurements, and h is the depth-of-focus of the laser beam. The average number of adsorbed molecules of Py per geometrical surface area unit participating in the SERS measurements (N surf ) was calculated assuming that the adsorbed molecules are spheres closely packed on a plane to form a hexagonal lattice. AFM measurements were performed to determine the geometrical surface area, see Figure 9a. More details can be found in the publication [15].
For the same amount of Ag deposit (0.02 mg/cm 2 , magnetron sputtering) on nanotubes fabricated at 10 V up to 25 V (see Figure 2), there was an increase in the SERS enhancement factor of from 10 5 to 10 6 (see Figure 9b). Figure 8. SERS spectra of pyridine adsorbed at the surface of nanotubes (10 V) annealed in air at 650°C for 2 h and coated with silver deposit: 0.01, 0.02, 0.03 mg/cm 2 . A reference spectrum for pure Ag (electrochemically roughened silver surface) is also given. Figure 9b is apparently due to a combination of two factors: a change in the specific surface area of the nanotubes with formation voltage [15], and a change in the distribution and size of the Ag nanoparticles with nanotube size [23]. These geometrical factors affect the properties of the silver "nanoresonators" produced on the tops and side walls of the TiO 2 nanotubes. The present results confirm the importance of the size of the geometrical surface area of the TiO 2 nanotubes for the plasmonic properties of the Ag-n deposit itself and, consequently, for the properties of the Ag/TiO 2 NT composite materials. Figure 10 shows SERS spectra of two other probe molecules: p-mercaptobenzoic acid (a) and DBRh dye (b) recorded on the platforms, which were characterized by an enhancement factor larger than 10 6 . In both cases, the spectra are of good quality and high intensity. They show that the background is not very high, and quite "flat" for both molecules. Our SERS experiments confirmed the good reproducibility of such substrates obtained by adorning TiO 2 nanotubes with Ag metal clusters and particles, which effectively support plasmon resonance [18]. 12 show R6G spectra taken with our Ag-n/TiO 2 NT platform covered with a solution containing 10 −7 or 10 −9 mol/l R6G. A rhodamine molecule is often used when the relationship between the local electromagnetic field enhancement and a large SERS signal is explored, which makes it possible to measure Raman spectra from a single molecule located on an Ag particle or individual Ag nanoparticles [36][37][38][39]. The characteristic peaks at ~970, 1150, 1220, 1300, 1340, 1410, 1500 and 1600 cm −1 correspond to the Raman lines for R6G [3,36]. In particular, the bands which are usually assigned to aromatic C-C stretching vibrations of R6G molecule are clearly visible. It can be seen that the SERS spectrum of R6G adsorbed on our Ag substrate exhibits enough intensity for this molecule to be detected even at a small concentration in an aqueous solution (below 10 −7 mol/l). As a result of this reduced concentration of R6G, some bands are suppressed (compare Figures 11 and 12). This sensitivity of our

Conclusions and outlook
PVD methods (the sputtering technique) provide a strictly controlled, stable distribution of Ag-n on nanotubular titania, thus offering suitable platforms for SERS investigations and for measurements of high-quality SERS spectra. Such spectra were obtained for pyridine, p-mercaptobenzoic acid, rhodamine and rhodamine 6G dye adsorbed on Ag-n-functionalized nanoporous layers of titania. For the SERS measurements (with R6G), excellent spectra were obtained, even for a concentration of R6G in a solution as low as 10 −9 mol/l. The E F value was found to be the largest (E F = 2.8 × 10 6 ) for the smallest nanotube diameter (d = 38 ± 9 nm). Stable, reproducible and highly active platforms for SERS investigations were obtained using relatively simple procedures and techniques.
The SERS enhancement induced by these new materials is associated primarily with two geometric factors: the specific surface area of the nanotubes, and an appropriate size and distribution of the Ag nanoparticles. These geometric factors (clearly visible using high-resolution microscopy) affect the properties of the metallic "nanoresonators" deposited on the TiO 2 nanotubes. Consequently, the probe molecules sense even slight changes on the surface, and these manifest themselves in the SERS intensity. However, the spectral differences observed result not only from the specific morphology, where the PVD techniques lead to the formation of cavities and slits that operate as adsorbed molecule resonators, but also from a specific interaction between the Ag nanoparticles and the TiO 2 nanotubes themselves. The further development of this type of SERS platform may involve using Ti of varying degrees of purity, and alloys of Ti, where the impurities and alloying elements will have an impact on the formation of nanoporous oxides layers by means of doping process (a change in the electron structure of the titanium oxide). As a consequence, this could lead to a significantly stronger enhancement of the intensity of the SERS spectra due to the occurrence of modified interactions between the doped titanium oxide and the metal nanoparticles, effectively supporting surface plasmons resonance (Ag, Au, Cu). All those factors should be taken into account when planning future analytical applications of new modified SERS substrates.