UNIVERZA V MARIBORU

FAKULTETA ZA NARAVOSLOVJE IN MATEMATIKO

Oddelek za fiziko

MAGISTRSKO DELO

Simon Hamler

Maribor, 2015

UNIVERZA V MARIBORU

FAKULTETA ZA NARAVOSLOVJE IN MATEMATIKO

Oddelek za fiziko

Magistrsko delo

VPLIV TEMPERATURE NA

POVRŠINSKO OJAČAN RAMANSKI SPEKTER

2,4,6-TRINITROTOLUENA

Master Thesis

INFLUENCE OF TEMPERATURE ON THE

SURFACE ENHANCED RAMAN SCATTERING

SPECTRA OF 2,4,6-TRINITROTOLUENE

Mentor: Kandidat:

doc. dr. Marko Jagodič Simon Hamler

Somentor:

dr. Hainer Wackerbarth

Maribor, 2015

ACKNOWLEDGEMENT

I would like to thank my mentor dr. Marko Jagodič and co-mentor dr. Hainer

Wackerbarth for help and guidance when writing this thesis. I would also like to thank

my family for always being there for me.

UNIVERZA V MARIBORU

FAKULTETA ZA NARAVOSLOVJE IN MATEMATIKO

IZJAVA

Podpisani Simon Hamler, rojen 4. 6. 1987, študent Fakultete za naravoslovje in

matematiko Univerze v Mariboru, študijskega programa Fizika, izjavljam, da je

magistrsko delo z naslovom Vpliv temperature na površinsko ojačan ramanski spekter

2,4,6-trinitrotoluena pri mentorju dr. Marku Jagodiču in somentorju dr. Hainerju

Wackerbarthu avtorsko delo. V magistrskem delu so uporabljeni viri in literatura

konkretno navedeni; teksti in druge oblike zapisov niso uporabljeni brez navedb

avtorjev.

Maribor, ______________ Podpis: ____________________

UNIVERZA V MARIBORU

FAKULTETA ZA NARAVOSLOVJE IN MATEMATIKO

Hamler S.: Vpliv temperature na površinsko ojačan ramanski spekter 2,4,6-

trinitrotoluena.

Magistrsko delo, Univerza v Mariboru, Fakulteta za naravoslovje in matematiko,

Oddelek za fiziko, 2015.

IZVLEČEK

Zaznavanje sledi eksplozivov, kot je trinitrotoluen (TNT), je pomembno področje pri

preprečevanju terorističnih napadov. Površinsko ojačana ramanska spektroskopija

(SERS) je postala močna detekcijska tehnika za identifikacijo majhnih količin analitov.

V magistrskem delu so predstavljeni podatki o TNT raztopini, naneseni na

nanostrukturirano zlato površino, ki je ogreta do 60 °C. Zaznane spremembe, ki jih

opazimo na podlagi mikroskopskih slik in SERS spektrov, razložimo s pomočjo

izhlapevanja, faznega prehoda in razgradnje TNT molekul. Vpliv temperaturne

odvisnosti na SERS učinek je bil raziskan na kemisorbiranem monosloju 4-

nitrothiophenol molekul. Da bi zmanjšali izhlapevanje TNT molekul, je bil med

plazmonsko površino in TNT vstavljen samosestavljiv monosloj mercaptoheksanola

(MCH).

KLJUČNE BESEDE: površinsko ojačana ramanska spektroskopija, eksplozivi,

temperaturna odvisnost, izhlapevanje, fazni prehod, molekulski razpad.

UNIVERZA V MARIBORU

FAKULTETA ZA NARAVOSLOVJE IN MATEMATIKO

Hamler S.: Influence of the Temperature on the Surface Enhanced Raman

Scattering Spectra of 2, 4, 6-Trinitrotoluene.

Master Thesis, University of Maribor, Faculty of Natural Sciences and

Mathematics, Department of Physics, 2015.

ABSTRACT

The detection of trace amounts of explosive like trinitrotoluene (TNT) is an important

issue in the prevention of terrorist attacks. Surface enhanced Raman scattering (SERS)

spectroscopy has become a powerful detection technique for identification of minute

amounts of analytes. This thesis presents data of TNT in solution, deposited on a

nanostructured gold surface, which is heated up to 60 °C. The observed changes in the

microscopy images and in the SERS spectra are explained by evaporation, phase

transition and decomposition of the TNT molecules. The impact of temperature

dependence of SERS effect is studied on a chemisorbed 4-Nitrothiophenol monolayer.

To minimize the evaporation of TNT molecules, a self-assembled monolayer of

mercaptohexanol (MCH) was inserted between plasmonic surface and TNT.

KEYWORDS: surface enhanced Raman spectroscopy, explosives, temperature

dependence, microscopy, evaporation, phase transition, decomposition.

CONTENTS

1 INTRODUCTION .................................................................................................. 1

2 RAMAN SPECTROSCOPY .................................................................................. 4

2.1 Energy units ..................................................................................................... 4

2.2 Degrees of freedom and molecular vibrations ................................................ 5

2.3 Basic theory ..................................................................................................... 7

2.3.1 Comparison of Raman and Fluorescence processes .............................. 12

2.4 Polarizability tensor ....................................................................................... 13

3 SURFACE ENHANCED RAMAN SCATTERING (SERS) .............................. 14

3.1 Electromagnetic Enhancement (EM) ............................................................ 15

3.1.1 Hot spots ................................................................................................ 21

3.2 Chemical mechanism .................................................................................... 21

4 EXPERIMENTAL SECTION .............................................................................. 23

4.1 Raman setup .................................................................................................. 23

4.2 Laser .............................................................................................................. 25

4.3 Spectrometer and CCD camera ..................................................................... 26

4.4 Plasmonic substrate ....................................................................................... 28

4.5 Microscope .................................................................................................... 28

4.6 Sample preparation ........................................................................................ 29

4.7 Data analysis ................................................................................................. 30

5 RESULTS AND DISCUSSION ........................................................................... 31

5.1 Microscopic observations .............................................................................. 31

5.2 SERS measurements of TNT ........................................................................ 32

5.2.1 Evaporation ............................................................................................ 35

5.2.2 Phase transition ...................................................................................... 35

5.2.3 Decomposition ....................................................................................... 36

5.2.4 Temperature dependence of the SERS effect ........................................ 37

5.3 TNT solution deposited on the substrate covered with mercaptohexanol

(MHC) monolayer .................................................................................................... 39

6 CONCLUSIONS .................................................................................................. 41

7 RAZŠIRJENI POVZETEK V SLOVENSKEM JEZIKU .................................... 43

REFERENCES ............................................................................................................ 46

1

1 INTRODUCTION

Owing to numerous attacks and attack attempts during the last years, the protection of

our society against terrorism has gained meaning. In this context, the detection of

explosives and their associated compounds is an important issue. This has led to

development of new detection technologies, especially in the field of homeland security,

to face the problems of hidden explosives at public places, such as airports, bus and

train stations. Many techniques have been investigated for this purpose. However, the

majority is not ideal for explosive detection, since they have disadvantages such as

invasiveness, detect only certain explosive, but fail to detect others, or require

complicated sample preparation. Vibrational spectroscopy has shown to be an excellent

technique for rapid, accurate quantitation and can be used for studying very wide range

of sample types and can be carried out from a simple identification test to an in-depth,

full spectrum, qualitative, and quantitative analysis [1].

Vibrational spectroscopy includes several different techniques. However, the most

important of them are infrared (IR) spectroscopy and Raman spectroscopy. Both of

these techniques can provide a complementary information about molecule vibrations

in many instances. Although both study the interaction of radiation with the molecule,

they differ in a manner in which photon is transferred to the molecule by changing its

vibrational state. IR spectroscopy measures transitions between molecular vibrational

energy levels, based on the direct absorption of light quanta. Absorption of photons

occurs, when the frequency of radiation by the polychromatic light matches that of a

vibration. Therefore, the molecule is prompted to a vibrational excited state. The loss

of this frequency of radiation from the beam after it passes through the sample is then

detected [1, 2]. On the other hand, Raman spectroscopy is based on a scattering

mechanism and requires monochromatic light for detection of molecular vibrations. A

portion of the incident photons will be scattered inelastically. Therefore, the energy of

scattered photons will differ from that of the incident photons. The energy difference

corresponds to the difference between the vibrational levels of the molecule.

The IR and Raman vibrational bands are characterized by their frequency (energy),

intensity (polar character or polarizability), and band shape (environment of bonds).

Since the vibrational energy levels are unique to each molecule, the IR and Raman

spectrum provide a ‘fingerprint’ of a particular molecule. The frequencies of these

molecular vibrations depend on the masses of the atoms, their geometric arrangement,

2

and the strength of their chemical bonds. Their spectrum provides information on a

molecular structure, dynamics, and environment [1]. In this thesis, the Raman

spectroscopy technique was used. There are two main advantages of Raman over IR

spectroscopy. The first one is that the samples can be confined or sealed in optical

transparent materials (glass, quartz), since they do not absorb the light. Because of this,

Raman spectroscopy is suited for the analysis of reactive or environmentally sensitive

compounds. The second advantage is that it is suitable for examining samples in

aqueous solution. Vibrations of water are weakly Raman active and hence does not

interfere the Raman signal, while in IR spectroscopy, the absorption of water is very

strong and thus often superimpose the signals of interest.

Raman spectroscopy is already an established technique in analytical and forensic

science, collecting a unique chemical signature of molecules. Almost all explosives can

be identified by their Raman spectra. Therefore, neat explosives have been extensively

studied by Raman spectroscopy [3–5]. Further advantages are that the detection is rapid

and non-invasive. There are hardly any limitations to the sample, which can be present

in different physical states or as a composition of different compounds. Moreover, there

is hardly sample preparation.

However, Raman spectroscopy is not suitable for the detection of trace amounts, as it is

needed for the prevention of bomb attacks. Nevertheless, the inherently weak Raman

process can be greatly improved using surface enhanced Raman scattering (SERS).

SERS combines low detection limits with high information content about molecular

identity making it highly suitable for trace analysis [6, 7]. Based on SERS single

molecule, detection was achieved. The enhancement factors can be as high as ~1014-

1015, if SERS is combined with other effects like resonance Raman. However, the

substantial contribution to the enhancement comes from SERS [8-11]. The surface

enhanced Raman effect is a product of two mechanisms, the electromagnetic and

chemical enhancement. The electromagnetic mechanism is believed to be responsible

for the bulk of the enhancement (~104-108) and is based on the increase of the

electromagnetic field strength in the vicinity of nanostructured metal surface. The

enhancement is greatest when the surface plasmon frequency and the incident light are

in resonance. In comparison to electromagnetic effect, the chemical enhancement is

quite modest (~10-102) and arises from interaction between adsorbed molecule and the

metal surface. The charge transfer between adsorbate and metal increases the

3

polarizability of adsorbed molecules. Another advantage of SERS is the quenching of

fluorescence, which is a known obstacle in Raman spectroscopy of explosives [3].

The aim of this work was to study how increasing temperature affects the SERS spectra

of TNT, deposited on a nanostructured gold surface and also if at the elevated

temperature, SERS measurements are still possible. Chapter 2 and 3 include necessary

theoretical background of normal Raman and surface enhanced Raman scattering. The

experimental setup is introduced in Chapter 4, with detailed description of its major

components. In the following chapter, we start with the microscopic observations of

TNT deposited on nanostructured gold substrate and continue with the investigation of

the temperature dependence of the intensity of TNT SERS spectra. Similar study is also

performed for the 4-Nitrothiophenol adsorbed on a substrate and mercaptohexanol

(MCH), which is placed between the surface and the TNT molecules. In conclusions,

we summarize our results.

4

2 RAMAN SPECTROSCOPY

The phenomena of inelastic scattering of light was first predicted by Smekal in 1923

and first experimentally observed in 1928 by C.V. Raman and K.S. Krishnan in India

and, independently, by L. Mandelstam and G. Landsberg in the former Soviet Union.

Since then, this phenomenon is referred as Raman effect. In the original experiment,

sunlight was focused onto the sample by the telescope and the second lens, which was

placed by the sample, collected the scattered light. Using the system of optical filters,

they showed the existence of scattered light with a frequency different from that of the

incident light – the basic characteristic of Raman spectroscopy [2].

2.1 Energy units

Light is an electromagnetic radiation that can act like waves or like a stream of small

“packets” of energy called photons. This is also known as wave-particle duality. Figure

1 illustrates a wave of linearly polarized electromagnetic radiation propagating in the x-

direction. It consist of electric and magnetic field components, which always oscillate

in phase with each other and are perpendicular to one another to the direction of wave

propagation. Since the Raman effect does not involve the magnetic component, only

the former will be further discussed. The oscillation of electric field of strength (E) at a

given time (t) is expressed as [12]:

0 cos 2E E t , (1)

where 0E is the amplitude of the incident electric field and is the frequency of the

radiation. In EM radiation, the frequency and the wavelength , are inversely

proportional to each other [12]:

c

, (2)

where c is speed of light. In Raman spectroscopy, instead of the wavelength the

wavenumber is used and given by:

7-1 10

cmnmc

. (3)

5

Figure 1: Linearly polarized electromagnetic radiation [13].

A transfer of energy from electromagnetic radiation to the molecule occurs when

following condition is satisfied:

cE h h hc

, (4)

where E is the energy difference between two quantized states and h is Planck

constant ( 346.626 10 Js ). Thus, wavenumber is directly proportional to the energy

of the transition. For example, 1 eV corresponds to 1240 nm, 142.4 10 Hz, and

8065 cm-1 [12].

2.2 Degrees of freedom and molecular vibrations

Degrees of freedom describes the motion of the atoms in x, y, or z direction. An N-atom

molecule will therefore have a total of 3N degrees of freedom of motion. Three of these

degrees of freedom describe the translational motion of the molecule and three of them

describe the rotational motion of the nonlinear molecule about the three principal axis

of rotation, which go through the centre of gravity. A linear molecule only has two

rotational degrees of freedom, since rotation around its own axis is not considered a

degree of freedom of motion (no nuclei displacements are involved). After subtracting

translational and rotational degrees of freedom from total 3N degrees of freedom, the

net vibrational degrees of freedom (number of normal vibrations) is 3 6N for

nonlinear and 3 5N for linear molecule. This means that for diatomic molecule, we

will have only one vibration. In the case of H2O molecule, we have 3 3 6 3 normal

vibrations as shown in Figure 2. These modes of vibration are symmetrical stretch,

bending, and asymmetric stretch. The linear CO2 molecule has 4 modes of vibration.

6

However, the model discussed here has only three. The fourth mode is also bending

vibration but in a different plane as shown in Figure 2. Such pair of vibrations with the

same frequency, different only in their direction, are called doubly degenerate vibrations

[2, 12, 14].

Figure 2: Normal modes of vibration for CO2 (+ and – denote vibrations going upward

and downward, in direction perpendicular to the paper) and H2O. Based on [12].

For better understanding of molecular vibrations, which are responsible for

characteristic bands in Raman spectra, we consider a simple model of diatomic

molecule, as shown on Figure 3. Atoms, with masses 1m and 2m are connected with

chemical bond, which in this case can be regarded as massless spring, with force

constant, k. Displacement of atoms from their equilibrium position is 1x and 2x .

Displacement of each of the two masses varies periodically over the period of time as a

sine (or cosine) function. Atoms oscillate with different amplitudes, but with the same

frequency, thus both masses go through equilibrium position simultaneously. The

classical vibrational frequency for diatomic molecule is:

1 2

1 1 1

2k

m m

. (5)

It can be seen from the above equation that the frequency of diatomic oscillator is a

function of atomic masses of the two atoms, involved in the vibration and the force

constant k, which is a measure of bond strength between the two atoms. If the atoms are

connected with double or triple bond, the force constant will be bigger and consequently

the frequency will also be higher [1, 12].

7

Figure 3: Motion of a simple diatomic molecule [1].

2.3 Basic theory

The Raman effect is a light scattering phenomenon. When a monochromatic light of

energy 0h interacts with the molecule in a material, it can be scattered. The oscillating

electric field of light distorts (polarize) the electron cloud around nuclei and form a

“virtual’’ state, which is not necessarily a true quantum state of the molecule. Because

the state is not stable, the photon is quickly re-radiated [15].

In vibrational spectroscopy, the detected energy changes are those that require changing

the vibration of nuclei. The dominant scattering, also called the Rayleigh scattering, is

a process where only electron cloud distortion is involved. This scattering is referred as

elastic scattering, since the energy/frequency of the photon is the same as before

interacting with the molecule. On the other hand, Raman scattering is a weak process,

where only one in 106-108 of scatter photons are Raman scattered. This occurs when

incident light induce a change in the nuclear motion and energy will be transferred either

from molecule to scattered photon or from incident photon to molecule. This process is

referred as inelastic scattering, since the energy of scattered photons differs from that

of the incident photons. If a molecule at the ground vibrational state is excited by the

incident photon to the virtual state and relaxes to a higher vibrational excited state, the

molecule gains energy and the energy of scattered photon is smaller than that of incident

photon 0 mh . This process is called Stokes scattering. If the molecule is already

in an excited vibrational state due to the thermal energy, the scattered photon may gain

energy from the molecule 0 mh , leading to anti-Stokes scattering. Since most of

8

the molecules at room temperature are in the ground vibrational state, the majority of

Raman scattering is Stokes scattering [2, 15].

Figure 4: Diagram for Rayleigh and Raman scattering [16].

The classical description of Raman scattering can be explained by Eq. (6). As already

mentioned earlier, the oscillating electric field of light E interacts with a molecule and

distorts electron cloud, thereby inducing an electric dipole moment P in the molecule.

The magnitude of induced dipole moment P depends on the polarizability of the

molecule and the strength of the electric field E of the incident radiation. This can be

expressed as:

0 0cos 2P E E t . (6)

Polarizability is proportionality constant and can be described as the ease with which

molecular orbitals are deformed, by the presence of the external field. The more easily

the electron cloud of molecule is distorted, the bigger the polarizability and thus greater

the induced dipole moment of the given field. If the molecule vibrates with a frequency

m , the nuclear displacements q can be written as

0 cos 2 mq q t (7)

where 0q is the vibrational amplitude. Using a small amplitude approximation,

polarizability can be expressed as a linear function of displacement in the form of Taylor

series:

9

0

0q

qq

(8)

Here 0 is the polarizability at the equilibrium position, and 0q

q

represents the

rate of change in polarizability with respect to the change in displacement from the

equilibrium position. If the derivative is equal to zero (no change in polarizability), the

vibration does not yield Raman scattering. Oscillations of polarizability cause the

induced dipole moment to oscillate at frequencies other than the incident frequency 0.

Combining Eq. (6) with Eq. (7) and Eq. (8), we obtain [12,15]:

0 0

0 0 0 0 0

0

0 0 0 0 0 0

0

0 0 0 0 0 0 0

0

cos 2

cos 2 cos 2

cos 2 cos 2 cos 2

1cos 2 cos 2 cos 2

2

q

m

q

m m

q

P E t

E t qE tq

E t q E t tq

E t q E t tq

(9)

The trigonometric identity 1

cos cos cos cos2

was used in the

final step of Eq. (9). According to classical theory, Eq. (9) demonstrates that the light

will be scattered at three different frequencies. The first term is the Rayleigh scattering

and represents an oscillating dipole which radiates light at the same frequency as the

incident light 0 . The second term corresponds to Raman scattering where oscillating

dipole radiates light at frequencies, which are different from the frequency of incident

beam that is 0 m (anti-Stokes) and 0 m (Stokes). The magnitude of these shifts

reflects the characteristic vibration of the molecule [12]. Some conclusions can be made

from Eq. (9):

1) As already stated before, if 0

0q

q

, the second term vanishes. The vibration

is not Raman active, since the molecular polarizability does not change during the

vibration.

2) If the vibration does not greatly change the polarizability, then the polarizability

derivative will be near zero and the signal from Raman scattering will be low.

10

Scattering intensity is proportional to the square of the induced dipole moment P,

which is proportional to the square of the polarizability derivative 2

q .

3) The equation also shows two possibilities to increase the Raman intensity. The one

is from the molecules with the larger polarizability and the other one is the stronger

electric field experienced by the molecules [17].

Thus, in Raman spectroscopy we measure the shift of the vibrational frequency m

from the incident beam frequency 0 . A Raman spectrum consist of scattered intensity

plotted vs. frequency shift between incident and scattered photons. The frequency shift,

also called Raman shift , is defined as [15]:

1 1

incident scattered

E

hc

(10)

where E is the energy difference between initial and final vibrational state of the

molecule. Raman shift is independent of wavelength of the incident beam incident , since

if we change incident , the wavelength of the scattered photons scattered changes in such

a way, that the remains the same.

As already mentioned in the beginning of this chapter, there are always more molecules

on the ground vibrational state than in the excited vibrational state at the room

temperature. This is why Stokes lines are much stronger than anti-Stokes lines. The

ratio of Stokes and anti-Stokes intensities depends on the population in ground and

excited vibrational states and can be obtained from the Boltzmann distribution [15]:

4

0

4

0

expmAS m

S Bm

I h

I k T

(11)

where Bk is the Boltzmann constant 231.38 10 J K . Measurement of this ratio can

also be used for temperature measurements. Since peaks from both lines are positioned

symmetrically with respect to the Rayleigh peak. Usual Raman spectrometers only

acquire Stokes spectra. A typical Raman spectrum, in this case CCl4, is illustrated in

Figure 5.

11

Figure 5: Raman spectrum of CCl4 [12].

The intensity of Raman scattering IR is given by [1]:

2

4

0RI I N tq

(12)

where is the frequency of the incident radiation, I0 is the intensity of the incident

radiation, N is the number of scattered molecules, is the polarizability of molecules,

q is the vibrational amplitude, and t is the acquisition time.

The above expression shows that increasing laser flux power or using shorter

wavelength excitation gives us a higher Raman intensity. However, since the molecules

usually have a bigger absorption cross section at lower wavelengths towards UV, the

fluorescence, which is a competing process and millions of times more efficient than

the Raman effect, is also higher and can thus overwhelm the Raman signal. Because of

such an inequality in signal strength, even the trace quantities of fluorescent materials

can mask the Raman signal of high-concentration analyte. This is why we usually use

excitation at longer wavelengths. Therefore, the wavelength selection is a balance

between minimizing the fluorescence and maximizing the signal strength [18].

12

2.3.1 Comparison of Raman and Fluorescence processes

Both Raman scattering and fluorescence produce photons with the frequencies different

from that of the incident photon, however, they are fundamentally different from each

other:

Raman scattering in a molecule is an instantaneous event in which an incoming

photon from the laser at 0 excites a molecular vibration m while emitting a

scattered photon at s o m . The incident photon does not need to be absorbed

and induce electronic transitions in the molecule, since Raman process can be

considered as an interaction with the ‘virtual state’, as depicted in Figure 4. Because

of this, Raman effects can take place at any frequency of the incident light, whereas

fluorescence is anchored at a specific excitation frequency.

Fluorescence, on the other hand is not an instantaneous, but a stepwise process. The

initial step involves the absorption of the incident light, where the system is

transferred from the ground single state S0 to a state in the vibrational substructure

of the first singlet state S1. The absorption process is shown in Figure 6a. Unlike in

Raman, the photon must have enough energy to reach S1 and start fluorescence

event. Once in the excited state, the molecule undergoes a series of vibrational

relaxations process, reaching the vibrational ground state of S1 (Figure 6b). After

certain amount of time (typically around few nanoseconds), the molecule relaxes

back to the vibrational levels of the ground state, thus emitting the photon (Figure

6(c)). In fluorescence, the emission process is completely independent of the initial

absorption, since both photons are not linked to each other in coherent and

instantaneous way like in Raman, where for each photon ‘taken’ from the laser,

there will be a scattered photon (one cannot exist without the other). However, in

fluorescence, there are situations where some potentially emitted photons from the

ground state of S1 go ‘missing’ (e.g. in non-radiative combination). Once the

molecule is excited to the S1 state, the best-case scenario is to ‘recover’ all the

photons that have been excited in the initial absorption process. However, small

fraction will usually be missing through a process that allows the molecule to relax

back to the ground state of S0 without emitting a photon. Therefore, the two

processes are effectively ‘disconnected’ in fluorescence (unlike in Raman) [19].

13

a) b) c)

Figure 6: Schematic presentation of fluorescence process as a sequence of events over

time. a) Fluorescence starts with the absorption of a photon. b) In the first electronically

excited state S1, the molecule undergoes vibrational relaxation and c) after few

nanoseconds, it relaxes back to the vibrational levels of the ground state S0, and thereby

emits a photon [19].

2.4 Polarizability tensor

To discuss Raman activity, we have to look more carefully at the polarizability . In

actual molecules, a nice linear relationship P E does not hold, since molecular

response to the applied electric field is not the same in every direction. Both P and E

are vectors, consisting of three components in x, y and z direction. Thus, Eq. (6) can be

written in the matrix form [12]:

x xx xy xz x

y yx yy yz y

z zx zy zz z

P E

P E

P E

. (13)

The first matrix on the right side is the polarizability tensor of second order. The tensor

is symmetric. The Raman scattering occurs when one of the components in

polarizability tensor changes during the vibration. For small molecules, it is easy to see

whether polarizability changes during the vibration. If we consider diatomic molecules

(e.g. H2) or linear molecules (e.g. CO2), the electrons are more polarizable (larger )

along the chemical bond than in direction perpendicular to it. Figure 7a shows changes

in polarizability from the vibrations of the CO2 molecule. Polarizability tensor is

graphically represented as the polarizability ellipsoid. This is a three-dimensional body,

whose distance from the electrical centre of the molecule is proportional to 1i, where

i is the polarizability in i-direction from the centre of gravity in all directions. When

14

xx yy zz polarizability ellipsoid will be a sphere and molecule is said to be

isotropic. For a completely anisotropic molecule, xx yy zz applies. The vibration

is Raman-active if the polarizability ellipsoid changes in its size, shape or orientation

and the intensity will depend on extent of this change. The 1 vibration is Raman-active

since polarizability changes in all directions. On the other hand vibrations 2 and 3

are Raman-inactive. Although in both cases the polarizability changes during the

vibration, the size and shape of the ellipsoid at +q and –q are identical by symmetry.

Note that the Raman activity is determined by the slope near the equilibrium position,

0q

q

(Figure 7b) [12].

a) b)

Figure 7: a) Changes in polarizability ellipsoid during three normal vibrations of CO2

molecule. b) The polarizability of CO2 as a function of displacement coordinate q for

1 and 3 vibrations (the function of the displacement is the same for 2 and 3 ) [12].

3 SURFACE ENHANCED RAMAN SCATTERING (SERS)

Surface enhanced Raman scattering (SERS) was discovered by Fleischmann and co-

workers in 1974 when they obtained an unusually strong Raman signal from pyridine

adsorbed on electrochemical roughened silver electrode. The reason to roughen the

electrode was to increase the surface area and thus the number of adsorbed molecules.

In 1977, Jeanmarie et al. and Creighton et al. confirmed the results and pointed out that

the Raman signal of molecules adsorbed on metal was enhanced by a factor of ~106,

15

compared to a signal from molecules in absence of metal. They reported that such

enhancement cannot be explained just by an increase in surface alone, but is also related

to an intrinsic surface enhancement effect. 40 years since discovery, improvements in

instrumental capabilities, better understanding of the enhancement effect and advances

in nanotechnology, made SERS spectroscopy a powerful analytical tool, used in various

fields, including physics, chemistry, and biology. It exploits the interaction of light,

molecules and metal nanostructured surface to enhance the Raman signal, in some cases

even to 14 orders of magnitude, thus allowing detection of single molecules.

The total enhancement is a product of two mechanisms, electromagnetic and chemical

or electronic enhancement. The dominant effect is electromagnetic enhancement (~104-

108, depending on the nanostructured surface) and is associated with magnification of

both incident and Raman-scattered fields, while chemical enhancement (≤102) arises

from electronic interaction between metal and adsorbed molecules. Both mechanisms

will be discussed in detail in the next chapter [20].

3.1 Electromagnetic Enhancement (EM)

The electromagnetic enhancement effect occurs at the metal-air interface. When

electromagnetic wave interacts with the metal surface, it causes collective oscillations

of the conduction electrons in metal - surface plasmons (SP). A plasmon is a quantum

of plasma oscillation. The plasmon can be consider a quasiparticle since it arises from

the quantization of plasma oscillations, just as phonons are quantizations of mechanical

vibrations. Thus, plasmons are collective oscillations of the free electron gas density,

for example, at optical frequencies. Surface plasmons are those plasmons that are

confined to surfaces and that interact strongly with light resulting in a polariton. If the

incident light is in resonance with plasmon frequency, the electromagnetic field at the

surface is enhanced. The electric field of SP can be expressed as

0

x zi k x k z tE E e

(14)

with for 0z , for 0z and where x is direction of propagation parallel to the

surface, z direction perpendicular to the surface, xk and zk are wave vectors

components along the x- and z-axis and is the frequency of the longitudinal

oscillation. The electromagnetic field disappears at z and is the strongest when

0z , which is typical for surface waves.

16

The solution of the Maxwell equations for the electric field from Eq. (15) at the metal-

dielectric interference with dielectric constants M and D yields the dispersion relation

SP of SP [21]:

M DSPSP

M D

c k

, (15)

where SPk is the wavevector of SP. The dielectric constant of metals is expressed as a

complex value ' ''

M M Mi . The real part '

M is associated with the polarizability of

incident light and imaginary part ''

Mi with the absorption. The complex function is

usually frequency dependent. In order to achieve the enhancing effect of the plasmons,

the electric field of the incident photon must oscillate parallel to the plane of the

incidence, so that its components lies in the direction of SP propagation. For a resonant

coupling of SP and photons, the energy E and the momentum p k has to be

conserved. The wavevector component of incident light ,Ph xk depends on the incident

angle and can be described with the help of the dispersion relation of the incident

photon Ph Ph

D

c k

, where Phk is a wavevector of an incident photon with the

following equation [21]

, sinPhPh x Dk

c

(16).

For resonance, the conservation of energy is given when Ph SP and the

following equation applies

, sin M DPh x D SP

M D

k kc c

(17).

The Eq. (17) relationship is shown graphically in Figure 8 [21].

17

Figure 8: a) Wavevectors components of incident photon, Phk and surface plasmons,

SPk , along a smooth metal surface. b) Dispersion relation of incident photon and surface

plasmons [based on 21].

Figure 8b shows that SP dispersion relation curve never intersects with the dispersion

relation line of a light in air with a dielectric constant, 1D . Consequently, on a

smooth metal surface, SP cannot be excited directly by just free-space light. The SP in-

plane wavevector is greater than that of incident light and since the momentum must be

conserved, the SP cannot radiate to the surrounding media. To ‘turn’ a light line to the

point, where it intersects with dispersion relation curve of SP to get a resonant effect,

and thus enhancement, the dielectric constant of the surrounding medium has to be

bigger than 1. This is usually achieved using a coupling medium such as prism.

Another possibility for resonant excitation of SP is roughening (usually

nanostructuring) the metallic surface to get a grating. In this way, ,Ph xk is matched with

SPk , by increasing the parallel wavevector component of the incident light with the

wavevector of the grating ,ph xk . Dispersion relation in this case is fulfilled by the sum

, ,

2sin sinPh x ph x SPk k n k

c c a

(18)

where n is the integer and a is the grating constant. , 0ph xk gives no solution to

dispersion relation. This is shown schematically on a Figure 9 [21].

18

Figure 9: a) Wavevectors components of incident photon Phk and surface plasmons SPk

along a nanostructured metal surface. b) Dispersion relation of incident photon and

surface plasmons [based on 21].

Due to the direction of SP, modes involving changes in molecular polarizability with a

component along the surface are the most enhanced. The most often used metals in

SERS are Ag, Au and Cu, since those materials have a negative real and small positive

imaginary dielectric constant (proportional to the damping of surface plasmons). Also

these materials fulfill the resonance condition in the visible or NIR frequency range. SP

can either be propagating in the x- and y-direction (~10-100 µm) along the metal-

dielectric interface and decay evanescently in the z-direction (~200 nm), or can be

localized on spherical particle for example. In the latter case, we talk about localized

surface plasmons (LSP). Since both, SP and LSP, are sensitive to the surrounding

dielectric environment, both are also used for SERS sensing experiments [8, 20, 22].

Figure 10: Illustration of the a) SP and the b) LSP [8].

A simplified schematic diagram for understanding the concept of electromagnetic SERS

enhancement is shown in Figure 11. The metallic ‘nanostructure’ is a small sphere with

the dielectric constant in a surrounding medium with a dielectric constant 0 . Since

19

the radius of the sphere is much smaller than the wavelength of light 0.05r , the

electric field is uniform across the particle and the Rayleigh approximation can be used

[23].

Figure 11: Simple schematic diagram for understanding the concept of EM SERS

enhancement.

A molecule in the vicinity of the sphere (distance d) is exposed to a field EM, which is

the superposition of the incident field E0 and the field of a dipole Esp induced in the

metal sphere, therefore 0M spE E E . The magnitude of the dipolar field Esp is given

as [23]:

3

0

0

02sp

rE E

r d

(19).

The field enhancement factor A is the ratio of the field at the position of the

molecule and the incident field

3

0

0 02

ME rA

E r d

(20).

A is particularly strong when the real part of the denominator is zero (i.e., real part

of is equal to 02 ). Additionally, the imaginary part of the dielectric constant

needs to be small for a strong electromagnetic enhancement. This condition describes

the resonant excitation of surface plasmons of the metal [23].

In an analogous way to the incident light, the scattered Stokes/anti-Stokes is enhanced.

Taking into account enhancement of the incident and the Stokes light, the

electromagnetic enhancement factor SG for Raman signals can be written as:

20

2 2 122 2 0 0

0 02 2

L S

em S L S

L S

rG A A

r d

(21).

The above equation shows that the enhancement scales as the fourth power of the local

field of the metallic nanostructure and that is particularly strong when both incident

light is in resonance with the surface plasmons and the inelastically scattered light is

close to this resonance [23].

The Eq. (21) also indicates that electromagnetic SERS enhancement is a long range

effect, which means that the adsorbate is not required to be in direct contact with the

surface of the metal. Enhanced EM fields generated by the surface plasmon resonance

(SPR) enables the detection of molecules even few nanometers from the surface of the

substrate. Long range effect of EM mechanism differs from the chemical enhancement

mechanism, where molecules have to be in direct contact with the surface. The detection

of the molecules nearby to the surface, which are not necessarily bound to it, can be

very useful in SERS applications, since many analytes have low or no affinity to Ag or

Au. In such cases, the surface can be modified with adlayers to improve specificity of

the analytes. The field enhancement around metal sphere decays with the growing

distance, described by the decay of the field of a dipole over the distance 3

1 d , as

shown in Eq. (20), to the fourth power, resulting to the 12

1 d (see Eq. (21)) [8].

The power of SERS signal is proportional to the following parameters:

2 2 R

SERS L L S adsP N I A A (22).

The power of the Raman signal SERSP depends on the number of molecules N, laser

intensity IL, enhancement factors of excitation LA , the scattered field SA , and on

the Raman cross section of the adsorbed molecule R

ads [20].

The increase in Raman intensity when the molecule is adsorbed on a SERS active

substrate is described by the enhancement factor (EF). The average EF for a SERS

system, which is evaluated at the single excitation frequency and the same acquisition,

is given as

SERS surf

NRS vol

I NEF

I N

(23)

21

where SERSI is a surface-enhanced Raman intensity, surfN is the number of molecules

adsorbed to the metallic surface that contribute to the SERS signal, NRSI is the intensity

of normal Raman scattering and the volN is the number of molecules in the excitation

volume [24].

The SERS enhancement factor also strongly depends on the orientation of the adsorbed

molecules with the respect to the metal surface. Enhancements are stronger if the

vibrations of the adsorbate are parallel to metal surface.

3.1.1 Hot spots

Hot spots are highly localized regions of intense local field enhancement, which are

caused by local surface plasmon resonances (LSPR). Hot spots occur when intense

electromagnetic field of two nanostructures superimpose. Nanostructures can be

nanoparticles or structures with a gap in-between. This phenomenon is strongly

dependent on the excitation wavelength, particle size, shape, and separation as well as

arrangement with respect to the polarization direction of the incident light. Many

articles in literature describe hot spots created between two nearby nanoparticles (gap

junctions) that line up their induced field with the external field. In other words, the

incident field induces two in-phase dipoles along the direction of the incoming field. If

d a , where d is the distance between particles and a is a particle diameter, the near-

field interactions will dominate and fall according to 3d . Therefore, in order to create

extremely high field confinements (hot spots), it is important to produce very small (few

nanometers) gap junctions, i.e. d a . When optical excitation is localized in such

small area, extremely large electromagnetic SERS enhancement up to 1210 can be

generated, thus allowing observations of single molecules [23, 25].

3.2 Chemical mechanism

Chemical or electronic SERS effect is a common name for different mechanisms, which

require direct contact between molecule and metal surface. Early researchers found out

that the SERS intensities between molecules of CO and N2 differ by a factor of 200

under the same experimental conditions. This result was very hard to explain just with

electromagnetic mechanism, since the polarizabilities of the molecules are nearly

22

identical and even the most radical variation in orientation upon adsorption could not

produce such large differences [26].

Chemical enhancement mechanism can be explained by the electronic coupling

between metal and a molecule, adsorbed on the metal surface. This adsorbate-surface

formation produces an increase of Raman cross section of the adsorbed molecule,

compared to the cross section of molecules in ‘normal’ Raman experiment. Other

possible explanation involve resonance Raman effect, which can occur due to shifted

and broadened electronic levels in the adsorbed molecule (compared to the free one) or

due to the new electronic transition in metal-molecule system. The later occur through

photon driven charge transfer process (PDCT) between metal and adsorbates, which is

shown in Figure 12, where HOMO and LUMO denote for the highest occupied

molecular orbital and the lowest unoccupied molecular orbital of the adsorbate,

respectively. The energies of HOMO and LUMO are approximately symmetric relative

to the Fermi level of the metal. The whole process can be described by the following

four steps [23, 26]:

Step 1: An electron-hole pair of the metal is created by the incident photon with energy

0h and the electron is excited to the hot-electron state.

Step 2: So-called ‘hot’ electron tunnels into the LUMO of the adsorbed molecule,

generating a charge transfer excited state.

Step 3: ‘Hot’ electron tunnels from LUMO (with changed normal coordinates of some

internal molecular vibrations) back to the metal.

Step 4: The electron recombines with the hole created in the step 1, which leads to a

vibrationally excited neutral molecule and to a emission of a Raman shifted photon,

with the energy h .

23

Figure 12: Schematic diagram of the photon-driven charge transfer model for a

molecule adsorbed on a metal [27].

Chemical enhancement mechanism is a short-range effect (0.1–0.5 nm), limited only to

first layer of adsorbed molecules. It depends on the geometry of bonding, the adsorption

site and the energy levels of the adsorbate molecule. The contribution of chemical

enhancement to the SERS intensity is estimated to be approximately 10-102. However,

is generally agreed that the electromagnetic enhancement is significantly larger in

magnitude. Although the chemical enhancement is not the general mechanism and is

restricted only to specific adsorbate-metal systems, it can still provide us useful

information on chemisorption and hence interactions between adsorbate and metal [20].

SERS spectrum can show some deviations in relative intensities compared to a normal

Raman spectrum of the same molecule. Interactions between molecule and metal may

cause, that the Raman lines are slightly shifted in frequency and changed in line width

compared with a ‘free’ molecule. Despite small changes, which can occur in SERS

spectrum, it still provides us a very clear ‘fingerprint’ of the molecule [23].

4 EXPERIMENTAL SECTION

4.1 Raman setup

The Raman setup used in our experiments consists of four major components:

excitation source (laser), light focusing and collecting system (in our case a fiber optic

probe head), spectrometer and CCD detector. A schematic of the apparatus is shown in

Figure 13.

24

Figure 13: Schematic presentation of major components in our Raman setup [28].

The SERS spectra were collected with a standard system (Kaiser Optical System Inc.,

Ann Arbor, MI, USA); the 785 nm (linewidth 0.06 nm) GaAlAs diode laser (Invictus,

Kaiser Optical Systems, Inc.) beam was focused onto the sample. The excitation and

scattered light were guided onto/from the sample by a multimode optical fiber equipped

with the probe head. The incident power of the laser emission was about 100 mW at a

probe head for 5 s recording with 1 accumulation on the detector. The scattered light

was coupled into the optical fiber by a confocal aperture and guided to the spectrograph

(PhAT SystemTM, Kaiser Optical Systems Inc.), which uses Volume Phase Holographic

(VPH) transmission gratings to perform filtering and dispersion functions. Prior to

entering the spectrograph, the scattered light goes through a holographic notch filter,

which cuts off photons at the laser frequency (i.e. Rayleigh scattering). The diffracted

light was recorded with a CCD camera (iDus, Andor Tecnology plc.) with a spectral

resolution of 5 cm-1. The most important prerequisite when comparing Raman shifts is

the reproducibility (or repeatability) of the experiment. Kaiser Optical Systems provide

a Raman shift tolerance between ± 0.5 and ± 1.0 cm-1, the individual system

performance will not vary to this extent. Upon calibration, a system should yield Raman

shift values reproducible to ± 0.1 cm-1 [7].

25

Figure 14: Front view of the Raman spectrometer.

4.2 Laser

Lasers are ideal excitation sources for Raman spectroscopy, since they provide a

monochromatic light with narrow bandwidth and high intensities necessary for

generation of a sufficient amount of Raman scattered photons. In addition, laser beams

have a small spot diameters that can be further reduced using optical lenses (smallest

possible diameter is approximately equal to the laser wavelength) for higher photon flux

at the measurement zone. Since the scattering intensity scales with the fourth power of

laser frequency (Eq. (12)), the most logical thing for improving Raman sensitivity

would be to use highest possible frequency. However, the problem that arises with the

use of high frequencies (or short wavelengths) is the emergence of fluorescence, which

can cover the Raman signal. The presence of the fluorescence can be reduced by using

longer wavelengths. Nowadays, the most common light sources for Raman

spectroscopy are diode lasers operating at near-infrared (NIR) wavelength.

Fluorescence at such wavelengths is not completely absent, but it is significantly

suppressed. The Raman intensity is however weaker, since the energy of radiation is

lower and the fourth power law applies.

We used a continuous wave (cw), Invictus 785 nm NIR diode laser, with a maximum

output power of 450mW. It is rated as class IIIb laser, meaning, that eye damage can

occur upon direct exposure to the laser beam. This class applies for laser with no more

than 500 mW of radiant power. It uses external cavity design to provide a narrow

26

linewidth and excellent wavelength stability. The Invictus laser also has integrated

holographic bandpass filter, which rejects any spontaneous emission from the diode that

is not at the lasing wavelength [29].

The probe head uses non-contact optics, which are optimized for incident NIR radiation.

The working distance is 1 cm, while the aperture ratio is f/2.0 [30].

4.3 Spectrometer and CCD camera

The spectral separation of Raman scattered light was performed using PhAT SystemTM

spectrograph from Kaiser Optical Systems Inc. Instead of a classical surface relief

reflection grating (usually in Czerny-Turner design) (Figure 15a) as dispersive element,

the PhAT SystemTM spectrograph uses Volume Phase Holographic (VPH) transmission

grating to perform filtering and dispersion functions (Figure 15b). VPH grating is made

from a layer of transparent material, usually dichromated gelatin, which is sandwiched

between two layers of clear glass or fused silica. When the light passes through the

optical thin film that has a periodic differential hardness or refractive index, its phase is

modulated. Hence the term ‘Volume Phase’. This is the biggest difference in

comparison to conventional reflection gratings, where the phase of the incident light is

modulated by the depth of a surface relief patterns. As in the conventional reflection

gratings, the spectral dispersion or angular separation of wavelength components in

diffracted light is determined by the spatial frequency of the periodic structure [31].

a) b)

Figure 15: a) Classical surface relief reflection grating. b) VPH transmission grating

[31].

Spectrally separated light is collected with the iDus DU420-BRDD charge-coupled

device (CCD) camera, which consists of rectangular two-dimensional arrays of 1024 ×

27

256 photosensitive elements (pixels). The pixel site is 26 × 26 µm, while the image area

is 26.6 × 6.6 mm. For readout, the detector uses so-called ‘Full Vertical Binning (FVB)’

method. Collected signal is converted into a 16-bit grayscale image. The operation

temperature of the camera was at 66 °C.

The silica based CCD sensors of the DU420-BRDD CCD camera have the highest

quantum efficiency in the NIR region (Figure 16). Since we used lasers with an

excitation wavelength of 785 nm, our spectral range of interest ranges from 785-915

nm, which corresponds to approximately 1800 cm-1 (Eq. (10)). In this region, quantum

efficiencies from 50 to 90% can be achieved. Figure 16 also shows a decrease in

quantum efficiency for wavelengths above 750 nm. The reason for the decrease lies in

the wavelength dependant absorption of photons in silica, which is why excitation

wavelengths longer than 785 nm can be unfavourable for detection with a CCD camera,

since weak Raman signals cannot be detected [21].

The so called “deep depletion“ technologies enables high quantum efficiencies in the

NIR. Devices manufactured with this technology have thicker photosensitive silicon

layers, which offer longer absorption path to photons with longer wavelength and thus

increase the probability for creation of excited electron-hole pairs [21].

Figure 16: Quantum efficiency of iDUS DU420-BRDD CCD camera [adapted from

21].

28

4.4 Plasmonic substrate

All SERS spectra were recorded on a commercially available nanostructured gold

substrate (Klarite®, Renishaw Diagnostics). The size of the substrate is 6 mm 10 mm,

with an active area of 4 mm 4 mm. The active area of the substrate consists of gold-

coated periodic square lattice of inverted pyramid pits (~1.4 μm wide and ~1 μm deep),

shown on Figure 17. The pyramid pits were produced using conventional optical

lithography on a (100) oriented silicon wafer followed by an anisotropic chemical

etching [6]. The substrate was opened from a vacuum-sealed package just prior to

experiment, to prevent any possible surface contamination.

Figure 17: SERS substrate with visible active area (left) and scanning electron

microscope images of the nanostructured gold surface (middle), and inverted pyramid

pits (right) [6].

The substrate was heated with Peltier element, with the size of 25 × 25 mm and

maximum working temperature of 138 °C.

4.5 Microscope

TNT solution on the SERS substrate was examined by Carl Zeiss El-Einsatz Axioskop

microscope We used a 100× microscope objective, with the numerical aperture of 0.90

and dark field illumination technique. Images were taken by the Lumenera’s

INFINITY2-2 digital CCD camera.

29

Figure 18: Carl Zeiss El-Einsatz Axioskop microscope.

4.6 Sample preparation

In SERS measurements, it is essential that the molecules are delivered to close

proximity of a metal surface. The molecules were analysed as thin films by dropping a

small volume of solution on the substrate. We used TNT solved in methanol/acetonitrile

(1 mg/ml), which was purchased from AccuStandard, Inc. (New Haven, USA). TNT is

a nitroaromatic compound (Figure 19a), which is mostly used for military and industrial

explosives applications. The melting point of TNT is at 80 °C and is thus far below the

temperature at which it will spontaneously detonate. It is also relatively insensitive to

shock and friction and the explosive cannot be initiated without a detonator.

Self-assembled monolayers of 4-Nitrothiophenol (technical grade from Sigma-Aldrich)

were generated by soaking the substrates in a 13 mM nitrothiophenol / ethanol (p.a.

from Sigma-Aldrich) solution for 24 h (Figure 19b). Before SERS measurements, the

substrate was rinsed with ethanol and left to dry in air for 10 min.

In the same way, by soaking the substrate in a 1 mM mercaptohexanol (MCH) / ethanol

solution for 24 h, was also made a self-assembled monolayer of MCH (technical grade

from Sigma-Aldrich) (Figure 19c).

30

a) b) c)

Figure 19: a) Chemical structure of TNT, b) 4-Nitrothophenol and c) MCH adsorbed

on a gold surface [6].

4.7 Data analysis

When recording a Raman or SERS spectra, in addition to the signal from the sample we

also detect some background noise, which can alter the profile of Raman bands.

Background noise can emerge because of different reasons; from not-fully suppressed

Rayleigh light from the laser, fluorescence or stray light. Because of this, background

subtraction or so-called baseline correction (Figure 20) is performed for each TNT

SERS spectrum. To perform a baseline correction it is necessary to create a baseline

based on a recorded spectrum, which is then subtracted from the spectrum. The baseline

was calculated with the help of local and global minima. Here, spectrum is divided into

intervals and in each of these intervals local, and global minima are searched. Per

interval, we have one support point or a node. These nodes are connected together with

the cubic spline, resulting in a baseline, which can be seen as a red curve in the Figure

20. The baseline is then subtracted from the recorded spectrum and we get the correct

TNT spectrum without a background noise (blue line in Figure 20).

Baseline subtraction on all spectra was performed with Origin 9.0.

31

Figure 20: Principle of the baseline correction. From a recorder TNT spectrum (black)

we subtract baseline (red), calculated on the basis of a local on global minima. Result

is a TNT spectrum without a background noise (blue).

5 RESULTS AND DISCUSSION

5.1 Microscopic observations

TNT diluted in methanol/acetonitrile was dropped on a nanostructured gold surface.

Even at small volumes, the solvents spread across the surface and evaporate, leaving

molecules adsorbed on the surface. Before and after the SERS measurements, in which

the substrate was heated to 60 °C, we examined the nanostructured surface with the

microscope. One corner of the substrate was chosen to make sure we would observe the

same area during the experiments. Figure 21a shows the nanostructured surface with

inversed pyramid pits after depositing a drop of solution on it. The border to the

unstructured area is also clearly seen. At first, it appears that observed spheres on the

surface could be liquid bubbles of the solution, but after being stable for 30 minutes, we

can conclude that all volatile solvents already evaporated. We assume that the flattened

spheres are pure TNT crystals. This agrees quite well with the SERS measurements

recorded immediately after microscope observations, which did not show any signs of

32

methanol or acetonitrile in the spectra. The distribution of TNT crystals on this substrate

is heterogenic and the crystal sizes are in the range from ~2 µm to ~12 µm.

Figure 21b shows the microscope image after heating it up to 60 °C and then cooling it

back to 20 °C. Almost all of the bright spheres on a substrate have disappeared and dark

patches are seen on the gold surface, where the large bright spheres were located before.

The smaller bright spheres disappeared completely, indicating evaporation of the TNT

microcrystals.

Figure 21: Microscope images of a solution on the nanostructured gold substrate a)

before and b) after heating to 60 °C.

5.2 SERS measurements of TNT

After the microscope examination, we immediately started the SERS measurements.

The temperature dependence of the intensity of the TNT Raman bands was studied. We

started at 20 °C and continued to 60 °C in 5 °C intervals. The acquisition time was 5 s.

A typical SERS spectrum of TNT is shown on Figure 22. The characteristic bands of

TNT are in the range between 200-1800 cm-1. Moreover, we observed peaks in the

region around 3000 cm-1, which can be assigned to different C-H vibrations.

At each temperature, the SERS spectrum was obtained for three random spots on the

substrate surface (Figure 23). Different intensities of the TNT bands for different spots

can be explained by the heterogenic distribution of TNT crystals in the excitation focus

on the nanostructured surface. Each spectrum for a given temperature is the average of

three spectra at different locations on the surface.

33

Figure 22: SERS TNT spectrum at 20 °C.

Figure 23: Three SERS spectra of TNT at 20 °C, obtained at three random spots on the

substrate surface.

34

TNT can be easily identified by the vibrational modes at the following frequencies: 323

cm-1 (2,4,6 C-N in plane torsion, ring in-plane bend), 792 cm-1 (ring in-plane bend, C-

CH3 stretch), 824 cm-1 (nitro group scissoring mode), 1207 cm-1 (ring breathing), 1356

cm-1 (4-NO2 symmetric stretching, C-N stretch), 1542 cm-1 (NO2 asymmetric

stretching) and 1616 cm-1 (phenyl modes) [32].

TNT SERS spectra from 20 °C to 60 °C are shown in Figure 24. Due to the temperature

change of the SERS substrate, a red-shift of the position of the frequencies up to 12 cm-

1 arises between SERS spectra of 20 °C and 60 °C. In Figure 25, the behaviour of the

most dominant band of the TNT spectrum at 1356 -1cm (NO2 symmetric stretching

vibration) is shown. The intensity decreases by a factor of 5. In the following,

contributions of evaporation, phase transition, decomposition, and temperature

dependence of the SERS effect are discussed on the base of the microscopy and SERS

results.

Figure 24: SERS spectra of TNT from 20 °C to 60 °C in 5 °C interval.

35

Figure 25: Intensity of the dominant TNT band (1356 cm-1) at different temperatures.

5.2.1 Evaporation

Evaporation of TNT molecules from the surface is a major contribution for the decrease

of the signals, as indicated by the microscopic images, in which some of the shiny

spheroids are disappeared after the heating. However, evaporation could not explain

dark patches and the changes in the spectra.

5.2.2 Phase transition

The change of the shiny TNT crystals to dark patches (Fig. 21) can be explained by a

phase transition. The intensity of the band at 1356 cm-1 decreases between 20 and 60

°C (Figure 25). Above 35 °C the decrease in intensity is steeper than at lower

temperatures, whereas above 55 °C the intensity appears to be constant. The melting

point of bulk TNT is at 80 °C. However, the melting point is size-dependent. Therefore,

the drop of the melting point can be explained by the small size of the TNT crystals on

the surface [33]. Moreover, volatile solvents can also have some effect on crystallization

of TNT, since the crystals could be formed differently, which may affect their quality,

resulting also in a melting point depression. Considering the heterogenic size

distribution of TNT crystals and their size dependent melting points, the temperature

dependent behaviour of the SERS intensities resembles conceivably a sigmoidal

36

melting curve, supporting the phase transition. The change of the shiny crystals and the

sigmoidal curve shape of the temperature dependence indicate melting of the TNT

crystals upon the heating. What exactly are these dark patches and in which phase are

they, is unknown.

5.2.3 Decomposition

Decomposition of the molecules is an issue in SERS spectroscopy, in particular as the

laser beam is focused on a small area at the surface. However, we have not observed

decomposition of TNT on such a substrate at room temperature under these conditions

(laser power, acquisition time) before. After heating the substrate to 60 °C, we cooled

it back to 20 °C and recorded a spectrum. The comparison of SERS spectra at 20 °C

before and after heating to 60 °C is shown in Figure 26. The heating and recording of

SERS spectra result in a non-reversible chemical process.

Figure 26: Comparison of TNT spectra at 20 °C at the beginning of SERS measurements

(black) with spectra at 20 °C, after cooling it down from 60 °C (red).

Characteristic TNT bands at 323 cm-1, 792 cm-1, 824 cm-1, 1207 cm-1 and 1542 cm-1

have totally vanished. The intensity of the dominant band at 1356 cm-1 has dropped to

approximately 24% of the original value and has also shifted by 12 cm-1 to a lower

37

frequency. Moreover, the band at 1617 cm-1 is shifted to 1607 cm-1. In contrast, the band

at 1128 cm-1, which had a low intensity at the beginning of the measurements, increases

for about four times. This clearly indicates that we can observe newly generated

unknown chemical species. A simple elimination of a nitro group which would

decompose TNT to DNT could not be explanation, as DNT can be identified by two

characteristic bands at 834 cm-1 and 1327 cm-1 [34]. Consequently, it looks like that

melting of the crystals is followed by the decomposition of the TNT molecules by

heating the plasmonic surface.

5.2.4 Temperature dependence of the SERS effect

Temperature dependence of the SERS effect could be further explanation for the

decreasing intensities in the SERS spectra. Pang et al. have published a study about the

temperature dependence of the SERS effect [35]. These authors found a decrease of the

SERS effect with rising temperatures. From 15 K to 300 K the enhancement drops by a

factor of approximately 3. To verify the impact of the temperature dependence of the

SERS effect, we adsorbed 4-Nitrothiophenol on the surface. These molecules form a

covalent gold-sulfur bond with the nanostructured substrate leading to a defined and

stable monolayer. Thus, evaporation and phase transition effects should not be relevant.

Spectra were recorded from 20 °C to 80 °C in 5 °C intervals; spectra were taken at three

different positions on the surface. Figure 27 shows SERS spectra of 4-Nitrothiophenol

between 20 °C and 80 °C.

The characteristic bands of 4-Nitrothiophenol are at 1081 cm-1 (C─S stretching

vibration), 1343 cm-1 (N─O symmetric stretching mode) and 1570 cm-1 (C═C

stretching mode of benzyl ring) [36,37]. The position of the most dominant peak shifts

slightly from 1344 cm-1 to 1342 cm-1 (Figure 27 inset).

The temperature dependence of the intensity of this band is shown in Figure 28. The

behaviour can be described by a linear decrease (Figure 28 inset). The overall decrease

in intensity between 20 °C and 80 °C is approximately 11 %. Besides this small drop in

intensity, we cannot find any significant changes in the spectra. The small decrease

could nevertheless be a consequence of desorption of the molecules caused by the

heating. On the other side, the drop can also be caused by the temperature dependence

of the SERS effect. However, the effect is small compared to the drop of the TNT

intensities by heating. It may contribute to rather small extent to the decrease. Moreover,

38

we can conclude that the used SERS substrate is suited for applications up to 80 °C.

That means that detection by SERS at elevated temperature is possible.

Figure 27: SERS spectra of 4-Nitrothiophenol recorded at 20 °C (black), 50 °C (red)

and 80 °C (blue).

Figure 28: Intensity of the dominant 4-Nitrothiophenol band at 1343 cm-1.

39

5.3 TNT solution deposited on the substrate covered with

mercaptohexanol (MHC) monolayer

MCH was adsorbed on a gold surface, where the molecules formed a defined and stable

self-assembled monolayer, similar as with 4-Nitrothiophenol. Then we dropped the

TNT solution on top of the formed monolayer. The main idea of inserting a layer of

MCH between the gold surface and the TNT was to diminish the evaporation of the

TNT molecules by the formation of hydrogen bonds between hydroxyl group of MCH

and the nitro group of the TNT.

Before the SERS experiment, we have taken microscope images with just MCH

monolayer adsorbed on the surface. As seen on Figure 29a, the MCH monolayer is not

visible and we can see just a nanostructured gold surface with some impurities on it

(bright yellow dots). After measuring the spectra of the MCH at 20 °C, we dropped the

TNT solution on top of the monolayer (Figure 29b). In comparison to Figure 21a, there

are not visible any TNT crystals in the shape of the flattened spheres. Noticeable are

just dark patches on the surface. Since on the planar gold surface area, TNT crystals are

clearly seen, we can assume that the added monolayer of MCH had an effect on the

adsorption of the TNT molecules on the surface. The formation of dark patches was

also observed after heating the uncoated substrate. Obviously, the heating and the MCH

layer cause the formation of a non-crystal phase of TNT molecules on the surface. The

substrate was then heated to 60 °C. Figure 29c shows a nanostructured surface after

cooling it back to 20 °C. Dark patches that were visible before heating are now

disappeared, which indicates that the TNT molecules evaporate from the surface.

a) b) c)

Figure 29: Microscope images of a) MCH monolayer and b) MCH monolayer with

added TNT on it, before heating to 60 °C. c) MCH monolayer with added TNT after

cooling it back to 20 °C.

40

Figure 30 shows comparison of a spectrum of TNT dropped directly on the surface of

the substrate (red) and spectrum of TNT, dropped on the MCH monolayer (black). Both

are measured at 20 °C with acquisition time of 5 s. Intensity of the most dominant band

of TNT, which is dropped on the MCH, is lower by approximately factor of 6. Decrease

in intensity because of MCH layer was expected, as the SERS effect depends strongly

on the distance between the molecules and the surface. Moreover, the MCH layer effects

the position of the TNT main bands. These are slightly (0-2 cm-1) shifted to the lower

frequency, which indicate interaction between the TNT-Au surface and TNT-MCH

layer.

Figure 30: Comparison of TNT spectra, where TNT was dropped directly onto the

surface (red) and onto the MCH monolayer (black).

Figure 31 contains the spectra of MCH (red) and TNT deposited on MCH layer before

(black) and after (blue) heating to 60 °C. Acquisition time was 30 s. Interestingly, the

signal from MCH layer is completely hidden under both of TNT spectra, since its bands

are hardly visible in TNT spectra, before and after heating. The most dominant peak is

around 1100 cm-1 and is attributed to the C-C stretching vibration. MCH forms a stable

and defined monolayer, in which the molecules are adsorbed in almost perpendicular

orientation to the surface. This might explain the low intensity of the band, as vibrations

oriented perpendicular are less enhanced by the SERS effect in contrast to parallel

41

oriented vibrations. Developing this argument further, the TNT should lay flat on the

MCH layer. A flat orientation is also assumed for the uncoated nanostructured surface,

which is in line with a similar intensity distribution of the Raman bands.

In conclusion, MCH does not prevent evaporation of the TNT molecules. The intensity

of the TNT band decreased with increasing distance from the plasmonic surface caused

by the spacer MCH. However, the MCH layer changed the adsorption of the TNT

molecules.

Figure 31: SERS spectra of MCH monolayer (red) and TNT deposited on MCH layer

before (black) and after (blue) heating to 60 °C. All spectra were recorded with

acquisition time of 30 s at 785 nm excitation.

6 CONCLUSIONS

Detection of trace amount of explosive materials such as TNT has recently gained the

highest importance in homeland security, environmental safety, and protection. The

surface of enhanced Raman scattering has become powerful technique for trace analysis

with its high sensitivity. It exploits the interaction of light, molecules and metallic nano-

scale roughened substrate, which greatly enhance the Raman signal.

42

In this master thesis, we have studied the influence of heating the plasmonic substrate

on the SERS spectra of the adsorbed TNT molecules. TNT in solution was deposited

on a nanostructured gold surface. After evaporation of the solvent, small TNT crystals

were formed in the shape of the flattened spheres. After heating the substrate to 60 °C

a part of TNT crystals disappeared, which agrees with the intensity decrease of the most

dominant TNT band, indicating evaporation of TNT.

However, evaporation cannot explain all of our observations. The change of the shiny

spots to dark patches in the microscopic images and the sigmoidal decrease of SERS

intensities of the dominant TNT band indicate melting of the TNT crystals by heating.

Finally, the changes in the SERS spectrum at 60 °C to the spectrum at 20 °C cannot be

explained solely by evaporation and melting. Disappearance of characteristic TNT band

and appearance of new band also points to the decomposition of TNT to other chemical

species. Moreover, we have studied the temperature dependence of the SERS effect.

Therefore, 4-Nitrothiophenol was chemisorbed to the nanostructured substrate. The 4-

Nitrothiophenol SERS spectra showed only a small decrease of the intensities,

indicating that if ever, the decrease of the enhancement up to 80 °C is small. In order to

reduce the evaporation of the TNT from the substrate, we immobilized a self-assembled

monolayer of mercaptohexanol (MCH) on the plasmonic surface. The MCH should

bind the TNT molecules stronger to the substrate. We indeed find a change in the

adsorption. The TNT does not form crystals on the MCH layer and the SERS signals

of TNT molecules are significantly reduced. However, upon heating the substrate the

SERS signal decrease again significantly, indicating that MCH cannot prevent the

evaporation of the TNT molecules.

The study shows that SERS at slightly elevated temperatures is still possible. The

enhancement of the plasmonic substrate is hardly affected up to 80 °C. However,

heating of small amounts of substance can lead to melting and decomposition. Even

when the bulk material is stable under such conditions. Explosive detection by SERS is

a field, in which these results should pave the way for new devices.

43

7 RAZŠIRJENI POVZETEK V SLOVENSKEM JEZIKU

Z naraščajočim številom terorističnih napadov v zadnjih letih je detekcija eksplozivov

in njim sorodnih spojin postala zelo pomembno področje. Ramanska spektroskopija je

že uveljavljena metoda pri analitičnih in forenzičnih raziskavah, saj je večino

eksplozivov mogoče identificirati na podlagi njihovih ramanskih spektrov, ki

predstavljajo kemijski ''podpis'' molekul.

Ramansko sipanje je neelastično sipanje svetlobe oziroma fotonov. Ko monokromatska

svetloba z energijo 0h interagira z molekulami v snovi, se lahko siplje. Pri tem

molekula iz osnovnega energijskega stanja preide v virtualno (navidezno) stanje, ki ni

pravo kvanto stanje molekule, zato se le-ta hitro vrne v (največkrat) osnovno stanje, pri

čemer emitira (sipa) foton. Običajno je frekvenca sipanega fotona enaka frekvenci

vpadnega, zato govorimo o elastičnem ali Rayleighovem sipanju, kjer se vibracijska

energija molekule ne spremeni. Pri majhnem deležu sipanih fotonov (en na 10

milijonov) pa je energija različna od vpadne. V tem primeru govorimo o neelastičnem

sipanju. Če molekula iz virtualnega stanja ne pade nazaj na osnovno stanje, ampak

zasede višje vibracijsko stanje, govorimo o Stokesovem sipanju. Vibracijska energija

molekule se poveča za razliko energije vpadnega in sipanega fotona 0 mh . V

primeru, da je pred sipanjem molekula že v vzbujenem vibracijskem stanju, sipani foton

pridobi energijo molekule 0 mh , kar vodi do anti-Stokesovega sipanja. Gledano

iz vidika klasične mehanike, nihajoče električno polje svetlobe deformira elektronski

oblak okoli jedra molekule, s čimer se inducira električni dipolni moment v molekuli,

ki je enak produktu polarizibilnosti molekule in jakosti električnega polja.

Polarizabilnost je odvisna od medatomskih razdalj, ki pa se zaradi nihanja molekul

nenehno spreminjajo. Polarizabilnost tako periodično niha s frekvenco nihanja

molekule. Zaradi pospešenega gibanja elektronov nihajoči električni dipolni moment

seva. Tako dobimo svetlobo, katere energija oziroma frekvenca se razlikuje od

energije/frekvence vpadne svetlobe.

Zaradi šibkosti signala pa ramanska spekstroskopija ni primerna za detektiranje zelo

majhnih količin vzorcev. Ramanski proces lahko znatno ojačamo z uporabo metode

površinsko ojačenega ramanskega sipanja (ang. surface enhanced Raman scattering –

SERS) tako, da molekule nanesemo na hrapavo (običajno nanostrukturirano) kovinsko

površino. SERS je posledica dveh mehanizmov, elektromagnetnega in kemičnega.

44

Slednji izhaja iz elektronskih interakcij med kovino in adsorbirano molekulo (≤102).

Večinski delež pri ojačitvi prispeva elektromagnetni mehanizem (~104-108), ki je

povezan z ekscitacijo lokalnih površinskih plazmonov in s tem ojačitvijo tako vpadnega

kot ramansko sipajočega elektromagnetnega polja.

Uporabljen ramanski merilni sistem je bil sestavljen iz štirih glavnih komponent: laserja

(785 nm), optičnega vlakna za fokusiranje in zbiranje sipane svetlobe, spektrometra in

CCD detektorja. V magistrskem delu smo raziskovali vpliv temperature plasmonskega

substrata na SERS spekter adsorbiranih TNT molekul. TNT, razredčen v

metanolu/acetonitrilu smo s pipeto nanesli na nanostrukturirano pozlačeno površino. Po

izhlapetju topila so se na površini substrata formirali majhni kristali TNT-ja, v obliki

sploščenih kroglic. Pred in po koncu SERS meritev smo na enakem mestu na

nanostrukturirani površini izvedli mikroskopsko analizo. Po opravljenih SERS

meritvah, kjer smo segrevali substrat od 20 °C do 60 °C s 5 °C intervali, večina TNT

kristalov izgine. To se ujema z intenziteto najbolj dominantne ramanske črte TNT, ki

se zmanjša za faktor 5, kar nakazuje na izhlapevanje. Vendar pa samo izhlapevanje ne

razloži vseh naših opazovanj. Pojav temnih lis na mestih, kjer so prej bili svetleči kristali

na mikroskopskih slikah, ter sigmoidni padec SERS intenzitete dominante črte TNT

nam nakazuje tudi taljenje TNT kristalov. Čeprav je temperatura tališča TNT pri 80 °C,

pa je ta odvisna od količine oziroma velikosti snovi. Znižanje temperature tališča lahko

razložimo z majhnostjo kristalov TNT na površini. Spremembe v SERS spektru pri 60

°C v primerjavi s spektrom pri 20 °C, pa ne moremo razložiti zgolj s izhlapevanjem in

taljenjem. Razgradnja molekul pri SERS spektroskopiji je lahko težava, zlasti ker je

laserski žarek usmerjen na majhno območje na površini. Izginotje karakteristične TNT

črte in pojav nove črte jasno kaže na razgradnjo TNT v drugo kemično spojino. Prav

tako smo preučevali temperaturno odvisnost na SERS učinek, saj bi ta prav tako lahko

vplivala na znižanje intenzitete pri SERS spektrih. Tako smo na substrat adsorbirali 4-

nitrothiophenol. Te molekule tvorijo kovalentne vezi med zlato nanostrukturirano

površino in žveplovim atomom, kar vodi do stabilnega monosloja. V tem primeru

izhlapevanje in fazni prehod ne bi smela imeti vpliva. Substrat smo ogreli do 80 °C.

SERS spektri 4-Nitrothiophenola so nam pokazali le majhno izgubo intenzitete, ki je

približno 11 %. Da bi zmanjšali izhlapevanje TNT-ja, smo na substrat absorbirali

mercaptohexanol (MCH), ki tvori stabilen samosestavljiv monosloj, podobno kot 4-

Nitrothiophenol. Na monosloj smo nato kapnili TNT raztopino. S tem so se tvorile

vodikove vezi med hidroksilno skupino MCH in nitro skupino TNT. Na ta način naj bi

45

MCH vezal TNT molekule močneje k površini. Mikroskopska opazovanja pred SERS

meritvami so razkrila spremembo v adsorpciji (TNT molekule namreč niso tvorile več

kristalov na MCH sloju). Tudi SERS signal TNTja je bil znatno manjši, saj je SERS

učinek močno odvisen od razdalje med molekulami in površino. Vendar pa se je ob

segrevanju substrata SERS signal ponovno bistveno zmanjša, kar kaže na to, da MCH

ne more preprečiti izhlapevanje TNT molekul.

Pokazali smo, da so SERS meritve pri višjih temperaturah še vedno možne, saj je

ojačitveni učinek plasmonskega substrata skorajda nespremenjen vse do 80 °C. Vendar

pa lahko segrevanje majhnih količin snovi vodi do taljenja in razgradnje le-teh tudi v

primeru, če je večja količina te snovi stabilna pri enakih razmerah.

46

REFERENCES

[1] P. Larkin, Infrared and Raman spectroscopy: principles and spectral interpretation

(Elsevier, USA, 2011).

[2] E. Smith and G. Dent, Modern Raman Spectroscopy – A Practical Approach (John

Wiley & Sons, Great Britain, 2005).

[3] M. L. Lewis, I. R. Lewis, P. R. Griffiths, Raman spectrometry of explosives with a

no- moving-parts fiber coupled spectrometer: a comparison of excitation wavelength,

Vibr. Spectrosc. 38, 17–28 (2005).

[4] I. R. Lewis, N. W. Daniel Jr., N. C. Chaffin, P. R. Griffiths, M. W. Tungol, Raman

spectroscopic studies of explosive materials: towards a fieldable explosives detector,

Spectrochim. Acta, Part A 51, 1985–2000 (1995).

[5] F. T. Docherty, P. B. Monaghan, C. J. McHuge, D. Graham, W. E. Smith, J. M.

Cooper, Simultaneous multianalyte identification of molecular species involved in

terrorism using Raman spectroscopy, IEEE Sens. J. 5, 632–639 (2005).

[6] H. Wackerbarth, C. Salb, L. Gundrum, M. Niederkrüger, K. Christou, V.

Beushausen, W. Viöl, Detection of explosives based on surface-enhanced Raman

spectroscopy, Appl. Optics 49, 4362–4366 (2010).

[7] H. Wackerbarth, C. Lenth, S. Funke, L. Gundrum, F. Rotter, F. Büttner, J.

Hagemann, M. Wellhausen, U. Plachetka, C. Moormann, M. Strube, A. Walte, Surface

enhanced vibrational spectroscopy for the detection of explosives, Proc. SPIE, 8896,

889609 (2013).

[8] K. M. Kosuda, J. M. Bingham, K. L. Wustholz, R. P. Van Duyne, Nanostructures

and Surface-Enhanced Raman Spectroscopy. In D. L. Andrews, G. D. Scholes, G. P.

Wiederrecht (eds.), Comprehensive Nanoscience and Technology 3, 263–301 (2011).

[9] S. Zou, G. C. Shatz, Silver nanoparticle array structures that produce giant

enhancements in electromagnetic fields, Chem. Phys. Lett. 403, 62–67 (2005).

[10] S. M. Nie, S. R. Emory, Probing single molecules and single nanoparticles by

surface-enhanced Raman scattering, Science 275, 1102–1106 (1997).

[11] K. Kneipp, H. Kneipp, I. Itzkan, R. R. Dasari, M. S. Feld, Ultrasensitive Chemical

Analysis by Raman Spectroscopy, Chem. Rev. 99, 2957–2975 (1999).

[12] J. R. Ferraro, K. Nakamoto, C. W. Brown, Introductory Raman Spectroscopy (2nd

ed.) (Elsevier, USA, 2003).

[13] Center for Materials and Devices for Information Technology Research

(CMDITR) Photonics Wiki, Electromagnetic Radiation. Acquired 7 April 2014 from

http://photonicswiki.org/index.php?title=Electromagnetic_Radiation.

[14] N. B. Colthup, L. H. Daly, S. E. Wiberley, Introduction to Infrared and Raman

Spectroscopy, (Academic Press, USA, 1990).

[15] R. L. Mccreery, Raman Spectroscopy for Chemical Analysis (John Wiley & Sons,

USA, 2000).

[16] B&W Tek, Inc., Theory of Raman Scattering. Acquired 10 April 2014 from

http://bwtek.com/raman-theory-of-raman-scattering/.

47

[17] S. Guoguang, Surface-enhanced Raman Spectroscopy Investigation of Surfaces

and Interfaces in Thin Films on Metal, PhD dissertation (Faculty of Mechanical

Engineering at the Ruhr-University Bochum, Bochum, 2007).

[18] P. Matousek and M. D. Morris, Emerging Raman Applications and techniques in

Biomedical and Pharmaceutical Fields (Springer, Germany, 2010).

[19] S. Schlücker, Surface Enhanced Raman Spectroscopy – Analytical, Biophysical

and Life Science Applications (Wiley-VCH, Germany, 2011).

[20] K. Kneipp, Surface-enhanced Raman scattering, Physics Today Online 60 (11), 40

(2007).

[21] K. Christou, Markierungsfreie Proteinanalytik mit oberflächenverstärkter

Ramanspektroskopie, (Dissertation, Germany, 2009).

[22] T. Vo-Dinh, Surface-enhanced Raman spectroscopy using metallic

nanostructures, 17, 557–582 (1998).

[23] K. Kneipp, H. Kneipp, I. Itzkan, R. R. Dasari and M. S. Feld, Surface-enhanced

Raman scattering and biophysics, J. Phys.: Condens. Matter, 14, R597–R624 (2002).

[24] P.L. Stiles, J. A. Dieringer, N. C. Shah and R. P. Van Duyne, Surface-Enhanced

Raman Spectroscopy, Annu. Rev. Anal. Chem. 1 601–26 (2008).

[25] Silmeco ApS, Hot spots. Acquired. 18 June 2014 from

http://www.silmeco.com/knowledge-base/hot-spots.

[26] A. Campion and P. Kambhampati, Surface-enhanced Raman scattering, Chem.

Soc. Rev. 27, 241–250 (1998).

[27] Z. Q. Tian and B. Ren, Raman Spectroscopy of Electrode Surfaces, Encyclopedia

of Electrochemistry, Wiley & VCH 3, 575 (2003).

[28] Kaiser Optical Systems, Inc., Raman Spectroscopy-An Overview. Acquired 21

May 2014, from

http://www.kosi.com/resources/product_literature_1/rtnote1b/Kaiser_TN1101.pdf.

[29] Kaiser Optical Systems, Inc., Invictus 785nm NIR Laser. Acquired 6 December

2014 from http://www.kosi.com/na_en/products/raman-spectroscopy/lasers/invictus-

785nm-VIS-Laser.php.

[30] Kaiser Optical Systems, Inc., Non-Contact Optics. Acquired 8 December 2014

from

https://www.google.si/url?sa=t&rct=j&q=&esrc=s&source=web&cd=3&cad=rja&uac

t=8&ved=0CDAQFjAC&url=https%3A%2F%2Fetd.ohiolink.edu%2F!etd.send_file%

3Faccession%3Ducin1108838053%26disposition%3Dinline&ei=7JCFVPrcBNKvabif

gtgB&usg=AFQjCNGdQ_NLp1F10bi-

wEtN_0JujFBbMQ&sig2=CaaJvTkZvPhCfkWM3ucNaw.

[31] Kaiser Optical Systems, Inc., Introduction to Volume Phase Holographic (VPH)

Transmission Gratings and Applications. Acquired 7 May 2014 from

http://www.kosi.com/Holographic_Gratings/vph_ht_overview.php

[32] J. Clarkson, W. E. Smith, D. N. Batchelder, D. Alastair Smith, A. M. Coats, A

theoretical study of the structure and vibrations of 2,4,6-trinitrotolune, J. of Molecular

structure, 648, 203–214 (2003).

[33] X. Di, B. Xu, G. B. McKenna, The melting behavior of trinitrotoluene

nanoconfined in controlled pore glasses, J. Therm. Anal. Calorim. 113, 533–537

(2013).

48

[34] M. K. Oo Khaing, C. F. Chang, Y. Sun and X. Fan, Rapid, sensitive DNT vapor

detection with UV-assisted photo-chemically synthesized gold nanoparticle SERS

substrates Analyst, 136, 2811–2817 (2011).

[35] Y. S. Pang, H. J. Hwang, M. S. Kim, Reversible temperature dependence in

surface-enhanced Raman scattering of 1-propanethiol adsorbed on a silver island film,

J. Phys. Chem. 102, 7203–7209 (1998).

[36] B. Dong, Y. Fang, L. Xia, H. Xu, M. Sun, Is 4-nitrobenzenethiol converted to p,p

'-dimercaptoazobenzene or 4-aminothiophenol by surface photochemistry reaction?, J.

Raman Spectrosc. 42, 1205–1206 (2011).

[37] S. Funke, H. Wackerbarth, The role of the dielectric environment in surface

enhanced Raman scattering on the detection of a 4-Nitrothiophenol monolayer, J.

Raman Spectrosc. 44, 1010–1013 (2013).