**1. Introduction**

Raman spectroscopy is a technique based on measuring Raman scattering, i.e., inelastically scattered photons. It belongs to the techniques of testing the oscillatory spectra of materials. Thanks to its application, it is possible to obtain information about the type and structure of various chemical and biological compounds, including explosives, drugs, molecules of poisonous compounds, as well as bacteria, viruses, and even cancer cells. This technique has a lot of advantages, but the biggest disadvantage is the low sensitivity of the method, which can be solved by the use of surface-enhanced Raman spectroscopy (SERS). It increases the intensity of the Raman signal up to 1014 times compared to the signal received in a classic Raman experiment [1]. The first observations of the surface-amplified Raman signal for

pyridine were made on the rough silver surface as early as 1974 [2]; however, the authors did not recognize the observed phenomenon as an extraordinary amplification or phenomenon. Since the discovery of SERS in 1977 [3], interest in this research technique has grown exponentially.

SERS is a well-known technique for detecting trace amounts (or even individual molecules) of chemical and organic substances in real time, using a specific structural and vibration "fingerprint" of the analyte being tested. By using this technique, it is possible not only to efficiently qualitatively analyze the particles adsorbed on the surface of the substrate but also to obtain valuable information on the structure of the detected compounds. SERS can be used, among others, in medicine, pharmaceutical and food industry, internal security, the detection of terrorist threats, nanosensors, or environmental protection [1, 4].

SERS is a noninvasive technique; it can be used for solid, liquid, and gas samples and does not require sample preparation before measurement; and it does not require vacuum. In biological analysis, it is possible to work in vitro and in situ. Analyses can be carried out in a wide range of measurement conditions (temperature, pressure) and are short-lived (lasts for seconds); a small amount of the analyzed substance (up to one molecule) is sufficient. It is possible to detect many components of a sample at the same time because the spectra obtained are characterized by high resolution.

A few years ago, SERS measuring equipment (based on confocal microscope, monochromator, and detector—cooled silicon matrix) was expensive and has significant dimensions. Nowadays, there are more and more inexpensive, portable technical solutions with increasingly better parameters (although their spectral resolution is still relatively low). Despite the advantages, one should take into account the limitations of this research methodology: problems with quantitative analysis, problems with the determination of fluorescent materials, and the inability to determine metals and alloys, as well as the stability and repeatability of measurements. The latter one problem can be solved by improving the SERS substrates used.

SERS substrate can be classified by structure of substrate surface, fabrication method, or application. According to the used structure, the SERS substrates can be divided based on:


Generally, all fabrication methods of the SERS substrate production can be divided into four main categories [5]:


**61**

*An Overview of Anodic Oxides Derived Advanced Nanocomposites Substrate for Surface...*

The two first categories (1, 2) can be classified as a "bottom-up" approach, while the third (3) is typical "top-down" approach [5]. The fourth category (4) collects all new technologies, which present new proposal and are based on new ideas, not used traditionally. For example, when anodic aluminum oxide (AAO) is used as a template for nanofabrication of metallic nanostructures, one can classify that method to the third category (3), while AAO decorated by metallic nanoparticles and used as an active SERS substrate will be included in nonconventional method (4). In summary, the classification of the fourth category depends on what experimental actions are included there, so the decision whether it is "bottom-up" or "top-down"

a.Military utilization and internal security, e.g., detection and identification of

To describe SERS substrate, the following parameters have to be taken under consideration: enhancement factor of signal for a given analyte, repeatability of measurements, shelf life/durability, required excitation wavelength and laser power range, temperature, and humidity sensitivity. To define SERS substrate efficiencies, the specific application, where the substrate is planned to be used, should be included. For example, when we plan to use SERS in blue or green-orange region (cover visible and NIR light), we can take an Au or Ag nanocolloids, but for UV region we need to use Al nanoparticles [6]. Generally, for detection of microorganisms and living cells, the colloidal nanoparticles are typically preferred as SERS substrates. Also, for detection of specific sequence of DNA or RNA and Ag or Au nanoparticles, Raman-active dyes can be applied, while in detecting pancreatic cancer biomarkers, the SERS in body fluids is required [7, 8]. Detection of bacteria *E. coli* can be made on SERS substrate with gold nanoparticles [9]. For detection of explosive materials, traditionally silver- or gold-based nanostructure is typically used [10–12], but Cu-based nanoobjects fabricated by laser ablation were also tested [13]. Another important factor that influenced the Raman enhancement is the environment, in which the detected molecules are embedded, such as body fluid,

Enhancing Raman scattering over ordinary ones, spectrum registration con-

Generally, the Raman enhancement depends on electromagnetic and chemical amplification in the presence of metallic nanostructure (Ag, Au, Cu, Al, etc.). Chemical strengthening arises as a result of the overlapping of valence orbitals of the adsorbed molecule and the metal conductivity bands. Then a charge transfer from the adsorbate analyze to the metal (or vice versa) becomes possible. The strength of the SERS enhancement is strongly dependent on the shape and size as well as

enhancement is the resultant of two mechanisms: chemical strengthening and

or greater. It is assumed that the observed signal

*DOI: http://dx.doi.org/10.5772/intechopen.92811*

approach should be taken after the analysis of the method. SERS substrate can be divided according to application:

illicit or dangerous materials such as explosives

d.Biology

e.Environmental science

f. Single molecule detection

ethanol water, or other solvent.

ditions are on the order of 106

electromagnetic field amplification.

b.Quality control in the food and pharmaceutical industry

c.Medicine, e.g., detection of cancer cells or defects in DNA

#### *An Overview of Anodic Oxides Derived Advanced Nanocomposites Substrate for Surface... DOI: http://dx.doi.org/10.5772/intechopen.92811*

The two first categories (1, 2) can be classified as a "bottom-up" approach, while the third (3) is typical "top-down" approach [5]. The fourth category (4) collects all new technologies, which present new proposal and are based on new ideas, not used traditionally. For example, when anodic aluminum oxide (AAO) is used as a template for nanofabrication of metallic nanostructures, one can classify that method to the third category (3), while AAO decorated by metallic nanoparticles and used as an active SERS substrate will be included in nonconventional method (4). In summary, the classification of the fourth category depends on what experimental actions are included there, so the decision whether it is "bottom-up" or "top-down" approach should be taken after the analysis of the method.

SERS substrate can be divided according to application:


*Assorted Dimensional Reconfigurable Materials*

research technique has grown exponentially.

terized by high resolution.

divided based on:

c.Nanowires

d.Composites

tional (4)

b.Metal thin films

a.Metal nanoparticles (e.g., colloids)

divided into four main categories [5]:

c.Template-assisted methods (3)

pyridine were made on the rough silver surface as early as 1974 [2]; however, the authors did not recognize the observed phenomenon as an extraordinary amplification or phenomenon. Since the discovery of SERS in 1977 [3], interest in this

SERS is a well-known technique for detecting trace amounts (or even individual molecules) of chemical and organic substances in real time, using a specific structural and vibration "fingerprint" of the analyte being tested. By using this technique, it is possible not only to efficiently qualitatively analyze the particles adsorbed on the surface of the substrate but also to obtain valuable information on the structure of the detected compounds. SERS can be used, among others, in medicine, pharmaceutical and food industry, internal security, the detection of

SERS is a noninvasive technique; it can be used for solid, liquid, and gas samples

A few years ago, SERS measuring equipment (based on confocal microscope, monochromator, and detector—cooled silicon matrix) was expensive and has significant dimensions. Nowadays, there are more and more inexpensive, portable technical solutions with increasingly better parameters (although their spectral resolution is still relatively low). Despite the advantages, one should take into account the limitations of this research methodology: problems with quantitative analysis, problems with the determination of fluorescent materials, and the inability to determine metals and alloys, as well as the stability and repeatability of measurements. The latter one problem can be solved by improving the SERS substrates used. SERS substrate can be classified by structure of substrate surface, fabrication method, or application. According to the used structure, the SERS substrates can be

Generally, all fabrication methods of the SERS substrate production can be

a.Chemical synthesis (like synthesis of colloids by microwave heating method) (1)

b.Deterministic pattering (including all lithographic techniques, like electron

d.Other methods not mentioned above, which can be classified as nonconven-

beam lithography and focused ion beam fabrication) (2)

and does not require sample preparation before measurement; and it does not require vacuum. In biological analysis, it is possible to work in vitro and in situ. Analyses can be carried out in a wide range of measurement conditions (temperature, pressure) and are short-lived (lasts for seconds); a small amount of the analyzed substance (up to one molecule) is sufficient. It is possible to detect many components of a sample at the same time because the spectra obtained are charac-

terrorist threats, nanosensors, or environmental protection [1, 4].

**60**


To describe SERS substrate, the following parameters have to be taken under consideration: enhancement factor of signal for a given analyte, repeatability of measurements, shelf life/durability, required excitation wavelength and laser power range, temperature, and humidity sensitivity. To define SERS substrate efficiencies, the specific application, where the substrate is planned to be used, should be included. For example, when we plan to use SERS in blue or green-orange region (cover visible and NIR light), we can take an Au or Ag nanocolloids, but for UV region we need to use Al nanoparticles [6]. Generally, for detection of microorganisms and living cells, the colloidal nanoparticles are typically preferred as SERS substrates. Also, for detection of specific sequence of DNA or RNA and Ag or Au nanoparticles, Raman-active dyes can be applied, while in detecting pancreatic cancer biomarkers, the SERS in body fluids is required [7, 8]. Detection of bacteria *E. coli* can be made on SERS substrate with gold nanoparticles [9]. For detection of explosive materials, traditionally silver- or gold-based nanostructure is typically used [10–12], but Cu-based nanoobjects fabricated by laser ablation were also tested [13]. Another important factor that influenced the Raman enhancement is the environment, in which the detected molecules are embedded, such as body fluid, ethanol water, or other solvent.

Enhancing Raman scattering over ordinary ones, spectrum registration conditions are on the order of 106 or greater. It is assumed that the observed signal enhancement is the resultant of two mechanisms: chemical strengthening and electromagnetic field amplification.

Generally, the Raman enhancement depends on electromagnetic and chemical amplification in the presence of metallic nanostructure (Ag, Au, Cu, Al, etc.). Chemical strengthening arises as a result of the overlapping of valence orbitals of the adsorbed molecule and the metal conductivity bands. Then a charge transfer from the adsorbate analyze to the metal (or vice versa) becomes possible. The strength of the SERS enhancement is strongly dependent on the shape and size as well as

dielectric constant of the metal (plasmonic) nanoparticles on SERS substrate [14, 15], because this affected the ratio of absorption and scattering events. When the particles are too large, the excitation of nonradiative multipoles can appear, and a decrease in the overall efficiency of the enhancement may occur. On the other hand, too small particles lose their electrical conductance and did not enhance the field. When the particle size move toward few atoms, we do not have plasmon anymore at the surface, because we do not have large collection of electrons oscillated together. Each experiment has an ideal particle size and ideal surface thickness to achieve best performance [16]. The strength of chemical enhancement is much lower than electromagnetic ones, but it is very important in determination of spectral pattern of SERS spectra, i.e., Raman shift or intensity ratio [17]. When the interaction between molecule and the metal produces a metal-molecule charge transfer (CT) state and the Raman scattering is excited with a laser source in resonance with that state, some Raman modes can be strongly enhanced. In summary, the critical issue for the improvement of SERS sensitivity and reproducibility is the rational design of a stable and uniform SERS-active substrate, because the Raman enhancement is susceptible to the composition, size, and morphology of the substrate [18–20]. Higher SERS substrate surface area favors obtaining enhanced higher signal, and for this reason the work is underway on obtaining SERS substrates constructed of nanostructures coated with metallic plasmonic nanoobjects.

The high local electromagnetic field near plasmonic nanostructures, when the tested molecules are in their immediate vicinity, provides enhanced high Raman signal. The degree of electric field enhancement depends on the composition, morphology, and geometric parameters of surface nanostructures, as well as the amount of the so-called hot spots (regions of intense local field enhancement believed to be caused by local surface plasmon resonances (LSPR) [21]), and decreases sharply as the distance between the ground and the analyte under test increases above a dozen nm. There are a lot of publications described in detail in the LSPR phenomenon [22–28], which justified not taking up this issue in this chapter.

Plasmon hot spots spread over the entire surface of the SERS substrate to ensure high sensitivity and repeatability of spectroscopic measurements; however, several conditions must be filled: (1) homogeneous distribution of plasmon nanostructures on a large surface with a strictly controlled distance, which effectively produces hotspot points; (2) it is necessary to efficiently and homogeneously deposit the studied molecules on the entire SERS substrate [29].

The main features of both the abovementioned mechanisms of SERS enhancement are different and will be given below. Chemical reinforcement can be characterized as follow:


**63**

*An Overview of Anodic Oxides Derived Advanced Nanocomposites Substrate for Surface...*

• The field enhancement effect depends mainly on the optical properties of

• Field amplification increases the scattering intensity of Raman order of 104

• This enhancement is felt up to several dozen nm from the surface of metal

• The effect strongly depends on the size, shape, and roughness, the surface morphology of the metal, or the size of the aggregates of metal atoms.

substrates have to be time stable and mechanically durable and ensure repeatable analysis results. To realize this aim, the SERS substrates based on nanostructures, including metallic nanostructures, have to be developed. The main trends in the

• Nanostructures covered by metallic nanoobjects/plasmonic nanoparticles

Brief descriptions of conventional fabrication methods and new directions to production of nanostructures for application in SERS are presented as follows: In recent years ideas and attempts of using the anodic aluminum oxide and anodic titanium oxide as SERS substrate were made. The ideas can be divided into

1.Using AAO as a matrix for the nanofabrication of nanostructures (like

2.AAO coated by metals (mainly by silver, AAO/Ag) in many forms directly used

3.ATO coated by metals (Ag or Au) which can act as recyclable SERS-active

More detailed information about research of anodic oxides using as SERS

In this chapter, the main emphasis will be on a new type of SERS substrates based on anodic metal oxides, which is the perspective and future direction of SERS substrates. Generally, AAO and ATO consist of arrays of regularly arranged nanotubes/nanopores, which can be filled or covered by other materials. Moreover, ATO

nanodots or nanowires) which can act as SERS substrates

To be able to take full advantage of the possibilities of the SERS technique, and even expand its application, it is necessary to develop effective SERS substrates that

in

and larger. These SERS

Electromagnetic field enhancement can be characterized as follow:

relation to normal condition registration of Raman spectrum.

*DOI: http://dx.doi.org/10.5772/intechopen.92811*

metal and is the strongest for Ag, Au, and Cu

will provide Raman dissipation signal enhanced up to 106

matter of searching suitable nanostructures are as follow:

• Colloids

• Layers of metals

the following groups:

substrate

• Layers covered by nanoobjects

• Mix of form mentioned above

as the composite SERS substrate

substrate will be given in Part 4 of this chapter.

(range covers several atomic layers).

*An Overview of Anodic Oxides Derived Advanced Nanocomposites Substrate for Surface... DOI: http://dx.doi.org/10.5772/intechopen.92811*

Electromagnetic field enhancement can be characterized as follow:


To be able to take full advantage of the possibilities of the SERS technique, and even expand its application, it is necessary to develop effective SERS substrates that will provide Raman dissipation signal enhanced up to 106 and larger. These SERS substrates have to be time stable and mechanically durable and ensure repeatable analysis results. To realize this aim, the SERS substrates based on nanostructures, including metallic nanostructures, have to be developed. The main trends in the matter of searching suitable nanostructures are as follow:

• Colloids

*Assorted Dimensional Reconfigurable Materials*

coated with metallic plasmonic nanoobjects.

[22–28], which justified not taking up this issue in this chapter.

studied molecules on the entire SERS substrate [29].

dielectric constant of the metal (plasmonic) nanoparticles on SERS substrate [14, 15], because this affected the ratio of absorption and scattering events. When the particles are too large, the excitation of nonradiative multipoles can appear, and a decrease in the overall efficiency of the enhancement may occur. On the other hand, too small particles lose their electrical conductance and did not enhance the field. When the particle size move toward few atoms, we do not have plasmon anymore at the surface, because we do not have large collection of electrons oscillated together. Each experiment has an ideal particle size and ideal surface thickness to achieve best performance [16]. The strength of chemical enhancement is much lower than electromagnetic ones, but it is very important in determination of spectral pattern of SERS spectra, i.e., Raman shift or intensity ratio [17]. When the interaction between molecule and the metal produces a metal-molecule charge transfer (CT) state and the Raman scattering is excited with a laser source in resonance with that state, some Raman modes can be strongly enhanced. In summary, the critical issue for the improvement of SERS sensitivity and reproducibility is the rational design of a stable and uniform SERS-active substrate, because the Raman enhancement is susceptible to the composition, size, and morphology of the substrate [18–20]. Higher SERS substrate surface area favors obtaining enhanced higher signal, and for this reason the work is underway on obtaining SERS substrates constructed of nanostructures

The high local electromagnetic field near plasmonic nanostructures, when the tested molecules are in their immediate vicinity, provides enhanced high Raman signal. The degree of electric field enhancement depends on the composition, morphology, and geometric parameters of surface nanostructures, as well as the amount of the so-called hot spots (regions of intense local field enhancement believed to be caused by local surface plasmon resonances (LSPR) [21]), and decreases sharply as the distance between the ground and the analyte under test increases above a dozen nm. There are a lot of publications described in detail in the LSPR phenomenon

Plasmon hot spots spread over the entire surface of the SERS substrate to ensure high sensitivity and repeatability of spectroscopic measurements; however, several conditions must be filled: (1) homogeneous distribution of plasmon nanostructures on a large surface with a strictly controlled distance, which effectively produces hotspot points; (2) it is necessary to efficiently and homogeneously deposit the

The main features of both the abovementioned mechanisms of SERS enhancement are different and will be given below. Chemical reinforcement can be charac-

• The enhancement of Raman scattered light intensity in relation to the usual

• Enhancement occurs only for particles that interact directly with the metal—as a consequence the range is limited to the monolayer adsorbate (high surface

• Chemical enhancing does not depend on the optical properties of the metal but on the nature of the metal-adsorbate interactions; for that reason they are also observed for other metals than traditionally used in SERS, like

• The amount of chemical enhancing depends on the location of the Fermi level of the metal and changes depending on the applied electrode potential.

.

Raman spectrum registration conditions is of the order of 102

**62**

terized as follow:

specificity).

Ag, Au, or Cu.


Brief descriptions of conventional fabrication methods and new directions to production of nanostructures for application in SERS are presented as follows:

In recent years ideas and attempts of using the anodic aluminum oxide and anodic titanium oxide as SERS substrate were made. The ideas can be divided into the following groups:


More detailed information about research of anodic oxides using as SERS substrate will be given in Part 4 of this chapter.

In this chapter, the main emphasis will be on a new type of SERS substrates based on anodic metal oxides, which is the perspective and future direction of SERS substrates. Generally, AAO and ATO consist of arrays of regularly arranged nanotubes/nanopores, which can be filled or covered by other materials. Moreover, ATO

after annealing can act as a semiconductor and be used, for example, as a photocatalyst for removal of residual particles left on the substrate after SERS measurements. Due to the listed above advantages, the anodic oxides in SERS applications become more and more popular but up to now only in the research area. It is worth to notice that aluminum and/or titanium anodic oxides as a SERS substrate are no commercially produced now, so as a consequence its fabrication methods can be classified as unconventional fabrication SERS substrate method. The possibilities of application of anodic aluminum oxide and anodic titanium oxide as SERS substrates will be described and discussed in details below.
