Fabrication and characterization of micro and nano-structures for surface-enhanced Raman spectroscopy

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Introduction

Nowadays the study of Surface-enhanced Raman scattering (SERS) eect involves many physics research groups worldwide. Since its rst discovery in the 1973 by Martin Fleischmann considerable improvements have been carried out. The prin-cipal mechanism proposed to explain the origin of SERS enhancement is named the electromagnetic mechanism. It is directly connected to the local electromag-netic eld enhancement due to the excitation of local surface plasmon resonances (LSPR) on metal substrates.

The main limit of the Raman scattering process is its very low eciency, and thus very low signal intensity detectable from small amount of material. The SERS eect allows to overcome this limitation, giving access to the prominent information contained in the Raman spectra, even in the case of very low concentration samples or very small amounts of material. This enables the distinction between similar molecules due to the identication of their specic nger-prints and the possibility to study the chemical and vibrational properties of a small amount of molecules located at a sub-micrometer scale near SERS active sites. All these properties have important applications, for instance, in the elds of biology (DNA recognition), analytical and applied chemistry (catalysis) and renewable energy (photovoltaics). Nanoscale features such as sharp points or interparticle junctions can generate intense electromagnetic elds that can amplify the eect of both the incoming ex-citation photons and the emitted Stokes shifted photons by orders of magnitude. These features must be well-controlled for SERS to be used eectively. Therefore, there is a strong interest about systematic studies reporting how the size, shape and interparticle distance of nanoscale metallic structures can improve SERS e-ciency. For this goal, and for future mass production applications, reliable methods to produce SERS substrates with suitable properties are strongly demanded. The activity of the present thesis, consists in the fabrication of metallic structures of dierent sizes and types, and the analysis of their enhanced SERS signals, with the aim to assess and improve Raman enhancement factors. The characterization of structural and SERS properties of the fabricated systems has been obtained by

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Atomic Force Microscopy (AFM), and Confocal Raman Microscopy (CRM) tech-niques, respectively. This work has been developed in collaboration between the Experimental Physics Department of Ulm University and the Physics Department of Pisa.

The SERS active systems produced and investigated in this thesis work are: (i) silver Fischer's patterns (FP) with lateral sizes of 1 μm, 300 and 100 nm, (ii) silver micro and nano-bowls (MNB) with diameters of 3, 0.9 and 0,4 μm, (iii) gold micro-cavities of 3 μm diameter with size-controlled clusters of nanoparticles (400 nm diameter) located inside them. In the rst two cases a well established production technique has been employed, the nanosphere-lithography (NSL), while in the case of the gold microcavities a novel fabrication process has been realized. The FPs are two dimensional (2-D) arranged triangular metal structures on a hexagonal pattern, while the MNB are mainly 3-D convex structures (i.e. 2-D lattices of overturned bowls) with the same periodical structure as above. The cavities are instead hemispherical metal holes, closely spaced but not adjacent. The FP structures are partially studied SERS emitters, but no systematic study of length scale dependence of SERS signal is present in literature. Concerning micro- and nano-bowls, just a few studies are present. The specic capability of the studied substrates to boost the Raman signal is quantied as the enhancement factor (EF), namely the ratio of the intensity of the enhanced Raman signal, over the signal measured on a reference sample. Such quantities are normalized by the number of involved Raman active molecules. At the state of the art, the Raman reference spectra are measured on the dye powder or in solution. This leads to arbitrary assumptions and drawbacks. In this thesis a new approach is proposed, adopting a dye single crystal of micrometer size as a reference. A careful analysis of the localized changes in Raman signal intensity over the surface of FP and MBN structures has been performed using statistical analysis methods and combining the CRM images with AFM morphology data. In the case of the micrometer size objects, the Raman active sites have been discriminated from the elastic scattering local maxima. The general behaviour in the case of the FP and MNB systems shows considerable SERS EF increase in the case of the smallest fabricated structures, in good agreement with the trend predicted by the theory. EF passes from the order of some thousands, as in case of the biggest structures, up to tens of millions, for the sub-micrometer structures.

Finally, a novel technique was used to fabricate gold micro-cavities containing clusters of a controlled size of gold nanoparticles. In fact, many experimental and theoretical studies have been carried out about the SERS signal from nanoparticles

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clusters, but no reliable production processes are available at the state of the art for fabrication of aggregates with specic and controlled characteristics. The prepa-ration method allowed the production of periodically arranged gold hemispherical cavities with the diameter of 3 μm, acting as to trap and conne on their bottom small clusters of gold nanoparticles of 400 nm diameter. As expected, the gold cavities themselves did not exhibit a signicant strongly SERS eect, but SERS ac-tive areas with repeatable size were detected inside the hemispherical metal holes with trapped particle clusters.

Thesis structure

This thesis is divided into four chapters. In the rst chapter, after mentioning some basic principles concerning Raman scattering, the physics involved in SERS is briey presented. The particular case of the resonant Raman scattering (RRS) is introduced. Then, the main contributions giving rise to SERS eect are described, with particular focus on electromagnetic enhancement mechanism. At the end of the chapter, the enhancement factor (EF) calculation is briey described.

Chapter 2 is divided in two parts. The rst one describes the methods used for the fabrication of the nanostructures presented in the thesis: (i) the nanosphere-lithography (NSL), a well established production technique, (ii) a novel fabrication process allowing the production of gold microcavities with controlled nanoparticle clusters inside. The second part of Chapter 2 illustrates the two characterization techniques employed, namely the atomic force microscopy (AFM) and confocal Raman microscopy (CRM).

Chapter 3 describes the preliminary measurements necessary for the optimization of the experimental parameters for the SERS analysis, such as the laser power and the integration time for the measurement of the spectra. A calibration method to estimate the reference spectrum for EF calculation is also proposed.

Chapter 4 reports the analysis of the fabricated structures is presented. First the case of the FPs is described for every specic size. Then a comparison between the results is realized. The micro- and nanobowl SERS signal is analysed for dierent sizes. Finally, the gold cavities with and without gold nanoparticles inside are characterized, both by the structural and SERS point of view.

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Chapter 1 The physics of the surface-enhanced

Raman scattering

The surface-enhanced Raman scattering (SERS) is a complex phenomenon that involves both optical spectroscopy and plasmonics [1]. For describing the SERS, rst the Raman scattering has to be introduced, as well as the resonant Raman scattering, which sometimes can be involved in the phenomenon too. Then the theories presented nowadays for the explanation of the process are reported, de-scribing briey how the plasmonics is involved and what are the most important conditions to be fullled for an ecient SERS eect. The following paragraphs will present the discussed topics, giving a general picture of the physical background of SERS.

1.1 The Raman scattering

The Raman eect was discovered in 1923 by the indian physicist Chandrasekhara Venkata Raman. This phenomenon consists basically in an inelastic scattering of light due to the interaction of exciting photons with a molecular system. In a classical picture the phenomenon can be described by a primary incident electric eld that will modify the charge distribution of the illuminated molecule, inducing a dipole moment. The sum of the induced dipole moments will act as a macroscopic polarization, that is the source of the inelastic scattered light, namely the Raman signal [2, 3].

In a simplied classical picture, the induced dipole ~µ is linearly related to the external electric eld ~E and the specic molecule polarizabilty α, that expresses the capability of the driving eld to deform the electron density of the sample out

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of is equilibrium conguration.

~

µ = αE~ˆ (1.1)

The nuclear motion will modify the polarizability as the electron density adia-batically adjusts to the nuclear positions, to minimize the energy of the system. Therefore α will be function of each normal coordinate q. Assuming that the exciting electric eld is oscillating,

~

E(t) = ~E0cos(ω0t) (1.2)

and expanding the polarizability into a Taylor series around the equilibrium nuclear coordinate, the following relation results:

µ(t) ≈ α0E0cos(ω0t)+ 1 2 ∂α ∂q q0 q0E0cos[(ω0=ωq)t]+ 1 2 ∂α ∂q q0 q0E0cos[(ω0+ωq)t] (1.3) where µ(t) is the time-dependent induced dipole, α0 is the rst term of the polar-izability Taylor expansion, ωq are the characteristic frequencies of rotovibrational modes along each nuclear normal coordinate.

In the relation 1.3 the Taylor expansion of the polarizability has been truncated after the linear term in the mode displacement, leading to the so called electrical harmonic approximation. The formula 1.3 shows that the time-dependent induced dipole acts as a secondary source of scattered radiation. Taking into account this equation it follows that the rst term is related to the Rayleigh scattering, while the second and the third ones are responsible respectively for the so called Stokes and anti-Stokes scatterings. In fact the latter two terms oscillate with shifted frequencies with respect to the exciting one and contain information about the molecular system via their dependence on ωq. The anti-Stokes term is usually suppressed with respect to the Stokes one, due to the Boltzmann factor that de-scribes the population of the thermally excited vibrational states. Thus the Raman emission depends on the non-zero derivative of the electronic polarizability at the equilibrium geometry along the related normal coordinate. This leads to specic selection rules that can be easily derived in some easy cases of two or three atoms molecules, just by simple simmetry considerations [1, 3]. For example, the Raman scattering selection rules are in principle dierent from the infrared (IR) emis-sion ones, thus it may happen that some molecules are Raman active but not IR emitting or viceversa.

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In terms of inelastic emission intensity, the oscillating induced dipoles behave as antennas. A Hertzian dipole emitted power depends on the squared second derivative of the dipole moment. Because of the fact that the scattered intensity IR in the Raman process is proportional to the total power emitted by the above induced dipoles, the following dependence is derived:

IStokes∝ ∂α ∂q 2 q0 (ω0− ωq)4E02 (1.4)

where only the Stokes emission has been considered, since it is the signal considered in this thesis work. Thus the polarizability gradient is responsible for the Raman scattering intensity. The parameters∂α

∂q

qq

and ωq provide information about the illuminated molecules, while quantities ω0 and E02 inuence the observed intensi-ties. Figure 1.1 depicts schematically the mechanisms of Stokes and anti-Stokes emissions.

This proposed classical picture gives an immediate and intuitive description of the phenomenon. Nevertheless, the explanation of many specic experimental evidences requires a quantum mechanical perturbation theory approach to light-matter interactions, that can be found in the reference [2]. For example both the resonant Raman scattering (RRS) and the surface-enhanced Raman scattering (SERS) are not explained by the classical theory.

Virtual4 energy4 states Vibrational4 energy4states Infrared4 absorption Rayleigh4 scattering Stokes4 Raman4 scattering Anti-Stokes4 Raman4 scattering 0 1 2 3 4

Figure 1.1: Energy level diagram and dipole transitions, with comparison between the Raman Stokes and anti-Stokes mechanisms, the elastic scattering and IR. Adapted from [3].

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1.2 The resonant Raman scattering

The RRS involves specic congurations where the incident wavelength directly excites an electronic transition of the molecule. In fact, in this case not only inelastic scattering plays a role, but uorescence is observed too, as depicted in Figure 1.2.

Fluorescence is usually exceeding the weak Raman signal; nevertheless, if it is considerably shifted with respect to the Stokes scattering, both phenomena can be detected and singled out. A possible approach that describes this phenomenon is based on a classical treatment of the electric eld and on a quantum mechanical treatment of the molecules. The second-order perturbation theory is employed. A step by step treatment can be found in reference [1]. In this description what comes out is that the resonant Raman cross-section depends on the time-dependent overlap of the excited-state vibrational wavefunctions with the nal ground-state wavefunction. The electronically resonant photon, used as a pump, allows the transition from the initial ground state to the electronic excited state. In RRS only the vibrational modes that are able to couple with the electronic resonance are visible.

Figure 1.2: Energy level diagram and dipole transitions of a molecule. The Raman, resonant Raman and ourescence processes are illustrated, schematic picture from [1].

For this reason, this technique has the potential to study the structure of elec-tronically excited states, but on the other hand some vibrational modes can be excluded, leading to simpler Raman spectra. In fact, only the transitions according

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to the selection rules of the resonant Raman scattering will be strongy amplied and thus visible in the spectra. Furthermore, the resonant enhancement boosts the intensity of the scattered light up to several orders of magnitude, increasing the signal-to-noise ratio and decreasing the quantity of molecules required for detec-tion. This result has interesting applications in the elds of biology, biochemistry, medical research [1].

1.3 The surface-enhanced Raman scattering (SERS)

The rst experimental observation of SERS eect was in 1974, by Fleischmann and coworkers [4]. Since then, important improvements have been realized, both in the fundamentals and applications. Two main mechanisms have been proposed, namely the chemical and the electromagnetic one. The rst one has a minor role and includes several factors related to the interaction between the adsorbed molecule and the metal surface, including molecular orientation and possible chem-ical bonds or metal-molecule complex formation. The second one, that usually dominates (signal enhancement of about one million can be easily obtained), is directly connected to the locally enhanced electromagnetic elds at specic sites of micro and nanostructured metal substrates. Those structures can be chosen in order to present specic local surface plasmon resonances (LSPR) that can lead to considerably intense local electric elds. In the case that both a LSPR of the metal substrate and an electronic transition of the molecule are properly excited by the laser radiation, the so called surface-enhanced resonant Raman scattering (SERRS) occurs. In this case the emitted intensity is higher than in the simple SERS eect.

Many studies are still focused on the basic investigation of SERS, trying to check the theories and provide valid explanations to novel experimental evidences [5, 6]. In fact these enhancement mechanisms are still subject of discussions. Neverthe-less, thanks to advanced fabrication techniques, which allow the control of the ob-jects at the nanoscale, many devices are already built nowadays and many other promising analytical tools will be presented in the next years [7]. The possible applications move from the study of biological systems as DNA recognition and neurons networks analysis to renewable energy in terms of the local monitoring of catalytical processes or energy transfer in photovoltaics [8, 9]. Furthermore, in the case of considerable enhancements the limit of single molecule detection can be reached, with important consequences in chemistry research and in the fabrication of highly sensitive nanosensors [10]. From the beginning, SERS was

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observed on systems where molecules were absorbed on rough metal surfaces, and then on strongly curved surfaces as noble metal nanoparticles. Thus the placement of this research eld in the nanoscience branch is quite natural. In the following paragraphs the two enhancement mechanisms will be treated more in detail.

1.3.1 The chemical enhancement

The chemical enhancement may be considered as a surface-induced resonance Ra-man eect. It depends basically on the chemisorption of the molecules on the metal surface, their orientation and surface selection rules. The chemisorption in-uences the electronic structure of the substrate allowing for a resonant excitation of a surface-induced charge-transfer state [1]. A simpler mechanism is based on the formation of a surface complex from absorbed molecules on metal surface. In this system a charge-transfer from the conduction band of the metal to the molecular orbitals or viceversa is allowed. In fact in this picture, without the presence of the metal, the energy gap between the highest occupied molecular orbital (HOMO) and the lowest unoccupied molecular orbital (LUMO) is rather high, while the formation of the complex allows a charge-transfer process where the charges can be transfered from the HOMO to the LUMO thanks to the interaction with the metal, because of the fact that the Fermi level is located in between them, as presented in gure 1.3 [12].

Figure 1.3: Illustration of the charge-transfer process that leads to the chemical contribution to the SERS signal, reproduced from [12].

This process reduces the energy gap between bands and increases the excitation probabilities of the transition.

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1.3.2 The electromagnetic enhancement

The electromagnetic enhancement results from the excitation of localized surface plasmons resonances in the nanoscale metal objects which serve as SERS sub-strates. In order to have prominent local electric eld enhancements, these nanos-tructures have to be smaller than the light excitation wavelength. If the last condition is fullled, these structures may be considered as emitting dipoles, pro-viding strong localized elds. The SERS mechanism can be thought as a two step process. At the beginning the molecules absorbed on such a particular substrate experience an enhanced electromagnetic eld due to the presence of LSPR. Sub-sequently, when the Raman signal is emitted, the nanostructures act as antennas for the enhancement of the inelastic scattered light. Due to the nature of the pro-cess, in order to maximize its eciency, it is important to choose the most proper laser light and SERS substrates with respect to the specic analysed molecule [10]. It will be shown as this experimental parameters can play a fundamental role in SERS. Figure 1.4 depicts the basic mechanism that occurs when a molecule absorbed on a metal nanoparticles is illuminated.

Figure 1.4: Illustration of the origin of the electromagnetic enhancement of Ra-man scattering for molecules located in close proximity to a metal nanoparticle, reproduced from [1].

The exciting electric eld ~E0 irradiates both the nanoparticle, with a dielectric constant εi immersed in a physical environment with a dielectric constant ε0, and the absorbed molecule. The latter emits a primary inelastic signal that is the same that would be emitted in regular Raman scattering. The nanoparticle generates a strong localized electric eld ~ELM that typically decays in a range of few nanometers, since it is the local near eld at the particle surface. This is the reason why the molecule has to be located in close contact to the metal nanostructure. At

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this point the molecule emits a stronger Raman signal ~EDip, due to the presence of this intense local electric eld. In the second step of the SERS process, the Raman photons excite the nanoparticle, which in turn emits a eld ~ESC. Since the Raman signal emitted because of the direct laser light irradiation is negligible if compared to these other intensities, the total SERS signal detected ~ESERS may be written as the sum of the ~EDip and ~ESC. However, the last term usually dominates all the others. The electric eld ~ELM is amplied with respect to the laser exciting one ~E0 by the eld enhancement factor g. This parameter depends on the polarizability of the nanoparticle, and hence on the frequency of the exciting electric eld .

~

ELM = g ~E0 (1.5)

The Raman signal emitted by the molecules ~EDip depends both on ~ELM and on the Raman tensor αR. This last quantity is related to the vectorial nature of the elds involved, including wave and polarization vectors [5]. However in this description it will be simplied and considered as a scalar quantity, for the sake of simplicity, as

~

EDip ∝ αRE~LM ∝ αRg ~E0 (1.6)

The Raman signal emitted ~EDip by the molecule can be enhanced by the nanos-tructure by a factor g0_{, in general dierent by g and dependent on the Raman} emission frequency. The SERS emitted electric eld ~ESERS depends on both the enhancement mechanisms:

~

ESERS ∝ αRgg0E~0 (1.7)

The SERS intensity is proportional to the square modulus of ~ESERS , thus it has the following dependence:

ISERS ∝ |αR| 2

|gg0|2I0 (1.8)

where I0 is the laser incident intensity. For not too high Raman wavenumbers, where g ∼= g0 can be assumed, the SERS intensity will be proportional to the fourth power of the enhancement of the local near eld g.

|g|4 = ~ ELM ~ E0 4 = ~ ESC. ~ EDip 4 (1.9)

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The SERS enhancement G is dened as follows G = αR αR0 |gg0|2 (1.10)

where αR0is the Raman polarizability of the isolated molecule. The most relevant contribution to the SERS signal is due to the metal nanostructure light scatter-ing. Even if the SERS intensity depends on the fourth power of the local electric eld enhancement, SERS is a linear optical eect since it is proportional to the rst power of the exciting light I0. In the right member of the relation 1.10 the two presented factors are directly related to the chemical and electromagnetic en-hancements contributions respectively. In fact while the rst eect has an inuence mostly on the Raman cross section, the second is related to the electromagnetic mechanism of the phenomenon. Of course the presented Raman polarizability not only depends on the molecule, but on the metmolecule complex, where, as al-ready discussed, charge-transfer mechanisms can occur [12]. The contribution to the total enhancement in the case of non resonant SERS is usually a factor up to 102, thus non dominant with respect to the other mechanism [13]. In terms of electromagnetic enhancement, typically two quantities are considered, namely the average and the maximum enhancement. In the rst case the electric eld is averaged on the nanostructure surface; in case that near eld eects between close objects are involved, an active area is dened for the average eld calculation [14]. The maximum electric eld enhancement can be orders of magnitude bigger than the average one. Taking into account the simplied picture proposed above, SERS is a near-eld phenomenon, and the emitted radiation relaxes according to dipole-selection rules. However, depending on the type of the fabricated struc-tures, other eects may occur. For example, an important local electromagnetic enhancement is generated in systems of two or more metal nanostructures, in the free spaces between them. In the case of gaps between metal objects, smaller than the nanometer scale, quantum mechanical tunnelling eects take also place. In this case novel plasmonic regimes can be identied, as the charge transfer plas-mons, when a short contact occurs between the two structures, or the bonding dimer plasmons, an intermediate regime between the near eld and the contact conguration, in the range of Ångstrom [15]. These novel plasmonic behaviours are interesting for the fundamental electromagnetic research, but not so promising for SERS applications, since the required gap between the particles is smaller than the typical molecule size, for this reason no Raman active molecule can be positioned in that gap. In general even in regular nanometer size gaps among nanoparticles,

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the enhanced local electric eld changes considerably with the interparticle dis-tance [16]. The typical proper gap size is in the range below 10 nm. On the other hand the proper size of the metal nanoparticles is in the range of 10 to 100 nm [13]. The lower limit is xed by the average molecules magnitude, the upper one by the exciting wavelength. The fact that the local electric eld enhanced by the nanostructures decays within few nanometers xes an upper limit on the size of the SERS analysed molecules too. For bigger molecular compounds the recognition of some specic funtional groups is required, the so-called molecular nger-prints. In the case of bigger structures plasmonic nonradiative multipoles are excited [13]. Nevertheless, even in the case of relatively big metal structures, a spectroscopic signal enhancement can be detected. In this case the surface plasmon resonance is red shifted with respect to the case of nanoscale objects. For this reason, bigger structures are more interesting for infrared applications [14]. Illuminating these size metal objects with a visible light the enhancement can be related mostly to the chemical eect, or to the surface roughness. In this case in fact, the surface irregularities can be described as a grating that can play a role in the matching between the photon and the plasmon wavevectors.

Furthermore, when the metal structures are smaller than the indicated range, the pseudo bulk description that leads to the surface plasmon resonances is no longer valid, because of the so-called quantum-size eects.

1.3.3 The electromagnetic theory of SERS

It is possible to use a simple modied Drude model in order to illustrate some of the presented points. The polarizability α of a metal nanosphere with a dielectric function ε(λ), radius R, in vacuum can be described as follows

α = R3ε − 1

ε + 2 (1.11)

the dielectric function of a Drude metal, modied for interband transitions can be written as:

ε = εb+ 1 − ω2

p

ω2_{+ iωγ} (1.12)

where the εb is the contribution due to the interband transition and ωpis the metal plasmon resonance, which is related to the metal electron density. The parameter γ is the electronic-scattering rate, which is inversely proportional to the electronic mean-free-path and to the metal conductivity. Substituting the relation 1.12 in

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1.11 the following expression is derived α = R 3_(ε bω2=ω2p) + iωγεb [(εb+ 3)ω2=ωp2] + iωγ(εb+ 3) (1.13) for α given, the relation 1.13 exhibits a pole when the exciting frequency ω is equal to: ωR = ωp √ εb + 3 (1.14) and the width of that resonance is given by γ(εb+3). For an ecient SERS process the quality of the plasmonic resonance is relevant. When γ is large, it lowers the resonance quality, that brings to a lower SERS enhancement. Considerably high values of this parameter can be due both to poor conductivity of the metal, or, in the case of signicantly small metal objects, to the fact that the electronic scattering at the particles surfaces become the dominant process. Thus, in the case of a given metal structure, to the rst order, the SERS intensity will depend basically on the size of the nanostructure. The most proper structure size is the best compromise between the exciting wavelength and the typical electronic mean free path of the conduction electrons. Thus, the most eective size range, as already mentioned, lies between 10 and 100 nm. In the case of strongly anisotropic structures or systems constituted by more metal objects coupled by near eld interactions, the polarizability term α depends on the relative orientation of the exciting light polarization with respect to the nanostructures [13], being usually expressed as a tensor [5].

Figure 1.5: Illustration (reproduced from [13]) of the charge distribution depen-dence by the electric eld orientation. The presented structure is a metal dimer, while the point located between them represents a molecule.

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This dependence of the polarizability with respect to the light polarization orienta-tion in the case of near eld coupling eects can be explained by a simple picture that involves a metal dimer. In the system presented in Figure 1.5, when the electric eld is polarized parallel to the dimer axis, the proximity of the charges, induced by optical elds leads to large elds in the gap and hence sensed by the molecule. In the orthogonal polarization direction such phenomenon doesn't oc-cur. The region between the nanospheres is the so-called hot spot where the local enhanced electric eld is considerably higher than in the rest of the system. According to the electromagnetic theory of SERS, Raman active areas correspond to these hot spots, where the SERS signal is considerably stronger than the rest of the substrate. Due to this correspondence, the study of the Raman active sites is used for the spatial localization of the electromagnetic hot spots. The last ones can be studied by theoretical calculations and numerical simulations, based on dierent methods as nite element method (FEM), or the discrete dipole approximation (DDA). These calculations basically solve the Maxwell's equations in the case of specic metal objects, with given geometries and materials properties.

Figure 1.6: E-eld enhancement contours external to a dimer of Ag nanoparti-cles separated by 2 nm. In the 3-D plots, the axis perpendicular to the selected plane represents the amount of E-eld enhancement around the dimer. Illustration reproduced from [16].

By the experimental point of view, the absorption spectra of specic SERS sub-strates are measured and compared to the theoretical simulated extinction spectra [10]. An example of simulated electric eld distribution on a metal dimer structure

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[16] is reported in Figure 1.6. The red arrow in the picture shows the electric eld oscillation direction. From the calculation results presented in Figure 1.6 an elec-tromagnetic enhancement of roughly 106 _{is found, while a maximum enhancement} of 1010 _{is reached for a specic conguration [16]. Nevertheless, the intensity of} the local electric eld in systems constituted by two close particles decreases quite fastly with the increase of the interparticle distance, even by just one nanometer. For this reason the fabrication of such SERS active substrates requires extremely high precision fabrication techniques [13].

Once the distribution of the strong localized electric elds is dened, their location can be compared to the Raman active areas measured on the SERS substrates. Two main experimental techniques are adopted for this purpose, the near eld scanning optical microscope (SNOM) and the confocal Raman microscope (CRM). The rst technique collects the near eld signal, the second one the far eld. Disordered arrangements of nanostructured systems as nanoparticle clusters are studied as well [17]. These aggreagates have the advantage of being easy to fab-ricate, due to self-assembling properties of the nano and micro-scale objects. On the other hand, they are not so easy to simulate, due to the considerable number of constituting elements and the low control on their spatial positioning. In these systems, both the single element plasmonic eect and the interparticle junctions have to be taken into account.

Figure 1.7: Example of an absorption spectrum, modied from reference [18]. The red absorption band is related to gold nanocavities located in the space between cylindrical metal structures horizontally placed on the substrate surface, when the laser light polarization is oriented perpendicularly to the cylinder axes.

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be-tween particles, the extinction spectra may present more or less sharp absorption peaks, located in specic spectral ranges [17]. The nanoparticle clusters may be seen as systems constituted by several dimers. The junctions of these dimers may be oriented in dierent directions, depending on the arrangement of the parti-cles. Because of the polarization dependence of the interparticle enhanced eld in dimers, the nanospheres clusters may have particular polarization anisotropies [13]. Since the SERS eect is a complex phenomenon where dierent light scat-terers are involved, the proper matching of a number of elements is required. If a resonant Raman scattering is pursued, the wavelength of the excitation laser light has to be located in the spectral range of the electronic resonance of the molecule. However, regardless of that, in order to have an ecient SERS eect, the exciting light wavelength and the Raman emitted band have to be located on the active substrate LSPR. Indeed systematic measurements realized by the group of Prof. Van Duyne [10] show how the SERS intensity is strictly correlated to the discussed conguration. Most precisely, the peak extinction wavelength of the LSPR, λmax, has to be positioned near the mid-point between the energy of the laser excitation and the energy of the Raman-scattered photons [10]. The measured absorption spectra show that the deposition of specic molecules can shift the spectral position of the LSPR. In fact the absorbed molecules layer can be modeled as a dielectric material layer superimposed on the metal, which has an inuence on the plasmonic behaviour of the metal structures [10]. Another consequence of this mechanism is that peaks with a smaller Raman shift show a maximum enhancement closer in energy to the LSPR λmax with respect to a peak with a larger Raman shift. In other words, the relative Raman intensity of some bands could depend on plas-monic eect. Figure 1.7 depicts an example of a typical good conguration in such respect [18]. In the presented system the laser wavelength is located at a slightly higher frequency than the absorption band maximum λmax, while the Raman band is overlapped to it.

1.3.4 The Enhancement Factor (EF) calculation

At the state of the art, the most quantitative approach for the analysis of a SERS system is represented by the enhancement factors (EF) calculation and measure-ment. In the calculations the electromagnetic contribution is usually considered, since it is the prominent one. The most classical assumption regarding the cal-culation of the electric eld distribution is the quasistatic one, but this condition is fullled only for objects smaller than the exciting light wavelength. However, taking into account the limits of this method, the electromagnetic model is

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suc-cessful both in accounting for the main observations in SERS and as a predictive approach for the outcome of experiments. As already discussed, some novel ex-pressions derived from the quantum-mechanical nature of the problem are under development [13, 15].

Adopting the current theory, a description of SERS enhancement in terms of Green's function spectral theory of optical responses of nanosystems has been de-veloped in the last years [13]. The enhancement considered in this picture is only related to the electromagnetic contribution. The problem is solved in the qua-sistatic approximation, where the condition that the nanosystem is much smaller that the light wavelength is fullled. A complete description is available on refer-ence [13].

By the experimental point of view, several denitions of enhancement factors are available in literature. As already discussed, this parameter can be related to exact SERS conditions: the employed active substrate, the molecular analyte, the excitation wavelength. A discrepancy is present sometimes at the state of the art when EF is mentioned. This comes from the wide variability in the denition of the EF and the way it is estimated in practice. Some valid and alternative experimental denitions have been proposed by Le Ru [19]. By the experimental point of view it is dicult to give a single EF denition. In fact, there are many dierent situations that can arise in SERS, related to the signal emitted by a single or multiple molecules, from which the diculty to precisely extimate the number of emitting molecules, averaged over time, spatial distribution and orientation of the probe on the surface. The enhancement felt by a given molecule at a specic point depends generally upon the Raman tensor of the probe and its orientation respect to the electric eld in that specic place. Furthermore the SERS emission is related to the relative orientation of the active substrate with respect to the laser polarization direction [19]. The most commonly used denition of experimental EF is the following: EF = ISERS NSurf ∗ NV ol IREF (1.15) where NV ol is the average number of molecules in the scattering volume for the Raman non-SERS measurement, while NSurf is the average number of absorbed molecules in the scattering volume for the SERS experiment. As it is evident, the EF calculation in this case appears as the ratio of the Raman signals detected on the SERS active substrate over the Raman signal of a non-enhanced Raman sam-ple. The quantities are normalilzed to the respective extimated numbers of Raman

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emitting molecules. Thus this procedure requires the preparation of a reference sample, where the Raman signal is only related to the regular dye molecules emis-sion, without any plasmonic or chemical contribution. This sample is usually a low concentration solution where the Raman active molecules are dissolved, or an analyte solid powder located over a silicon substrate [20, 21]. Both these strategies have positive and negative characteristics that will be discussed in detail further on. Nonetheless, in this Thesis an original approach is proposed to overcome some of the common diculties (see section 3.2).

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Chapter 2 Experimental techniques of

fabrication and characterization of

SERS structures

2.1 Sample micro-nanofabrication

This chapter describes the two methods used for the samples preparation. All the samples have been prepared as a part of this thesis work, and with the support of researchers and technicians.The rst one, based on nanosphere-lithography [22, 23], is a well known technique already used at the state of the art of research. In this case it has been used for the production of Fischer's patterns and hemispherical micro-nanostructures.

The other method has been developed during this thesis experimental work, im-proving some pre-existent techniques and introducing new procedures [24, 25, 26]. It ordered 2D lattices of hemispherical metal cavities with controlled size and quite homogeneous metal layer, overcoming the current main limitations of the conven-tional electrochemical deposition [28]. At the end of the section 2.1.2 some possible perspectives for further improvement are presented.

The production of the samples, despite consisting in several steps, are quite fast and inexpensive because both techniques are based on micro-nanoparticle self-assembling processes. These methods allow the preparation of dierent sizes and metals structures, down to the dimension of 10−7 _{m. Most of the limitations} preventing a downscale of such procedures to smaller sizes are related to the degree of the order obtained by self-assembling the nanoparticles: the smaller the size of the objects to produce, the more dicult the control of structuring process.

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Once optimized, overcoming the current drawbacks and improving further every single step, these techniques can become very promising in the close future, even for possible mass production applications.

2.1.1 The nanosphere lithography

This production technique was developed by U. C. Fischer and H. P. Zingsheim [27] in the eighties and is nowadays a well established procedure for the generation of periodic structures on the submicron scale [22].

It is an appealing fabrication method because of its technical simplicity and exi-bility. It allows the production of two main kinds of structures: triangular shaped objects distribuited in hexagonal lattices, called Fischer's patterns, and hemispher-ical structures arranged according to two-dimensional (2-D) regular arrays. The size and the shape of both structures are related to the employed polymeric spheres and therefore easily controllable. The dimensions and distance between them are easily controlled by the template size [29].

The method basically consists in three subsequent steps (the third is not necessary for the micro- and nano-bowls case):

1. fabrication of a single monolayer of hexagonal close-packed polystyrene micro or nano particles absorbed on a substrate,

2. deposition of a metal layer on the spheres pattern, which acts as a mask, 3. removal of the particles by ultrasonication, which uncovers the underlying

Fischer's patterns.

The process is shown directly below in gure 2.1 :

(A) PS particles colloidal crystal (B) metal deposition

(C) sonication removal of covered particles

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In the following subsections the experimental process is presented in more details. Template mask preparation

Colloidal suspensions of polystyrene spheres in water have been employed. Parti-cles are well monodisperse, having a typical diameter variation of less than 1.5%. Samples with diameter size of 3 μm, 0.9 μm, 0.4 μm in water were used. Chemical reagents as surfactant are present in water in order to avoid cluster formation due to the hydrophobicity of the polymer.

Glass cover slides of 20x20 mm2_{and 24x24 mm}2 _{are employed as substrates. They} are etched in a plasma cleaner, for 7 seconds at a pressure of 0.5∼10−2 _{mbar in} order to increase the surfaces hydrophilicity and assure a proper spread of the water and particle suspension on the substrate. The employed plasma cleaner is a FEMTO, Diener electronic.

After this processing some drops of particle suspension are added to the glass supports with a micropipette and let dry lying horizontally at 25°C for 5 hours. Before casting, the liquid is sonicated in order to dissolve any particle agglom-eration which may be present. No gas ow has been applied during the drying process. The specimens are kept in a closed plastic box at this step in order to avoid air ows and temperature inhomogeneities.

At this step the solvent evaporation proceeds from the edge of the deposited drop to the center, determining the formation of a meniscus. The latter it retracts moving in a radial direction, and the liquid front retraction is responsible for the transportation process of the particles on the glass during sedimentation [31].

10 μm

(b) (a)

Figure 2.2: Optical pictures of (a) 0.9 and (b) 3 μm polystyrene particles close packages.

Depending on the quantity of suspension dropped with respect to the substrate surface and on the drying process conditions, mainly temperature and gas ow, the system can lead to the formation of single layers or multiple closed-packages, and/or even vacancies. The used solvent is relevant too, because of the surface

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tension involved. As shown in Figure 2.2 after drying the absorbed particles on the surface are arranged as close-packed islands.

10 μm

Figure 2.3: Optical picture of dislocations in the pattern, 0.9 μm diameter. Figure 2.3 shows the presence of some defects of the periodical structure, called dislocations. They result from the simultaneous growth of dierent small colloidal crystals. At the moment of the contact the dislocation elongates alongside one of the crystal directions [30]. These defects are mainly due to the diameter dispersion of the polymeric particles. The self-assembled ordered islands may have dierent sizes, in this case they are typically in the order of 50 μm.

When a lower density of spheres than Figure 2.3 is present, usually as a conse-quence of a too dilute suspension, these ordered clusters can be found as dendritic structures. In the case that bigger periodic arrays are required, the size of the defect-free areas can be increased optimising the experimental parameters. With this fabrication technique, extensions up to 1 cm2_{are reachable [32]. Anyway, the} typical overall size of ordered areas we obtained was more than sucient for the goals of the thesis.

Metal deposition and ultrasonication

A 60 nm silver layer is deposited on the two dimensional arrays of nano- or micro-spheres by thermal evaporation. The evaporation consists in a bell jar vacuum chamber equipped as follows. The vacuum system is composed by a rotary and a turbo-molecular pump, with an operation pressure of nearly 10-6_{mbar. The} evaporation system consists in a holder supporting the sample, a heater and an evaporation boat with the material to be evaporated, 22 cm under the sample. A quartz oscillator crystal is used for monitoring the thickness of the deposited silver layer. The optimal evaporation rate was chosen in the range of 5-20 Ås-1_. A schematic picture of the instrument is presented in Figure 2.4. The metal deposition is anisotropic because of the preferential metal atoms bombardment direction. Thus the obtained layer can be inhomogeneous in thickness. There are

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shadowing eects too, which are also responsible for the typical Fischer's pattern structures that will be shown in details in chapter 4. After this process all the sample surface is covered by the metal.

metal vapour

metal source

vacuum chambersubstrate holder with quartz oscilallator crystal

substrate

vacuum pumps evaporation boat with heater

Figure 2.4: The metal deposition apparatus.

On the top of the close-packed arrays of spheres there are hemispherical structures covered by silver, while on the glass there is just a at metal layer. The upper surface of the deposited metal layer has a characteristic roughness which depends on both the original one of the underlying glass substrate and on the deposition conditions.

The specimens are then sonicated in ethanol for 1 minute and washed three times with 1 ml of the same solvent. At the end they are dryed with a nitrogen ux. The sonication process produces the detachment of the silver coated polystyrene spheres, leaving the above mentioned Fischer's patterns on the surface. Figure 2.5 shows an overview of the produced stuctures by means of optical microscopy images. Optical microscope images allow, for the largest diameters, the hemispher-ical structures as well as the triangular shaped particles of the Fischer patterns to be well visible. Actually, the triangular units composing the Fischer patterns have concave sides and sharp corners, as it will be shown better in chapter 4 from a characterization of the structure by means of Confocal Raman Spectroscopy and Atomic Force Microscopy.

In the case of the intermediate structures it is dicult to distinguish the shape of the particles. Despite that, the hexagonal symmetry is still observable. In the Fischer's pattern structure (panel (d)) many dislocations are present, coming from defects of the polystyrene particles self-assembling. Finally, the optical observation of the smallest structures presented in Fig 2.5 (c-f) is not achievable, due to their size which is smaller than the microscope resolution. In spite of this, the presence

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of a regular pattern is perceptible. A more detailed description of the obtained structures can be found in chapter 4, thanks to the characterization by means of Confocal Raman Spectroscopy and Atomic Force Microscopy.

(b)

(a)

(d)

(c)

(f) (e)

Figure 2.5: Optical microscope images (100X magnication) of the fabricated stuctures from polystyrene particles of diameter 3 micron (rst row, panels a and b), 0.9 micron (second row, panels c and d), 0.4 micron (third row, panels e and f). On the left and right column hemispherical metal structures and Fischer's patterns, respectively, are shown for the dierent sizes.

2.1.2 Fabrication of microcavity localized nanoparticles

The aim of this fabrication method is to obtain ordered arrays of nanoparticle clusters localized in microcavities. The production is achieved following basically three steps:

1. formation of an ordered pattern of isolated polystyrene particles with a con-trolled distance between them,

2. production of metal hemispherical cavities, 3. insertion of nano-objects inside these holes.

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A further step consists in nano-objects insertion inside the cavities, that will be described later in this chapter .

The technique comes from the elaboration and improvement of some other partial procedures, developed mainly in the last two years [24, 25, 26, 33] along with the development of new processes. Despite on the fact that the presented method is based on several steps, once that the correct experimental parameters have been found the entire fabrication can be performed quite rapidly. All the production levels may be further optimized and improved if required, as well as dierent shapes and sizes of objects can be designed.

The schematic view of the complete technique is shown below in Figure 2.6. Figure (1a) and (1b) show the PDMS stamp with holes, respectively with and without polymer particles inside (1c) stands for the glass support with a thin polymethyl-methacrylate layer on top, (2) depicts the ordered polymer pattern obtained on the substrate. (3) represents the gold covered sample, (4) the previous element with glass and resin superimposed. Figure (5) shows the obtained metal cavities with the polymer spheres inside and (6) without them. The last step (7) represents the nano-objects inclusion.

(1a)

(1b)

(1c)

(2) (3) (4)

(5) ₍₆₎ ₍₇₎

Figure 2.6: Schematic description of the fabrication of microcavities and nanopar-ticles location at their bottoms.

Fabrication of ordered patterns of polymeric particles

The rst step of this method requires the production of a periodic array of polystyrene particles, that will be used afterwards as a template for the metal deposition. It is important to control the distance between the spheres, since a close-package,

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clearly easier to realize, would lead to the formation of interconnected cavities. Moreover in a close-package structure, depending on the metal deposition tech-nique, the material can penetrate more or less deeply into the structures, however, if the particles are in contact, such points will never be lled, leading to incomplete structures. On the other hand, a reasonable distance among the spheres can guar-antee the fabrication of hemispherical metal structures, or even quasi-spherical ones [26].

In order to arrange the particles in a controlled pattern on the substrate two dierent procedures are used in literature [25], both based on the employment of a PDMS (Polydimethylsiloxane) stamp with holes of a well dened diameter and distance. Such techniques consist basically in wet transfer mechanisms [25], that determine a controlled arrangement of the polymer spheres on a substrate. The latter is covered by a thin PMMA (Polymethyl methacrylate) layer, required for subsequent detachment of the glass support.

The PMMA layer formation The PMMA layer is deposited on the substrate by spin-coating. This technique consists in a rotating plate where the sample is xed. The rotating speed is controlled as well as the operating time. During the rotation of the substrate, the required solution is deposited on the sample. Due to centrifugal acceleration, the liquid material is distributed on the surface and the solvent is partially casted away or evaporated, depending on the rotating speed. Changing the experimental parameters the layer properties can be controlled. The employed spin coater is a WS-400-6NPP Spin Coater - Laurell Technologies. At the beginning the correct experimental parameters for the production of this deposition have been identied. The lm has to be homogeneous in thickness and smooth enough: irregularities have to be negligible if compared to the polymeric sphere particles, well below the micrometer scale. A PMMA (molecular weight of 14000 g/mol) 1% weight solution in methyl ethyl ketone has been employed. An amount of 60 μl of solution has been deposited by spin coating technique on a rotating clean 24X24 mm2 _{glass substrate. A WS-400-6NPP Spin Coater - Laurell} Technologies was used. The utilized spin coater speed is 4500 rpm. The spinning velocity aects both the lm thickness and the evaporation rate. A spinning time of 4 minutes was enough to obtain complete solvent evaporation. The spin-coater has to run untill the complete solvent evaporation, otherwise the formation of polymer grains may occur, even in the size of micrometers. Furthermore the migration of these particles on the surface due to centrifugal acceleration may lead to the formation of polymer empty channels on the glass. Such defects are shown in

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gure 2.7.

10 μm

10μm 10μm

(a) (b)

Figure 2.7: Optical pictures of a PMMA layer on the glass support with (a) grains, (b) grain with empty channel.

The PDMS stamp moulding The PDMS with hemispherical holes stamp is fabricated moulding it on a master Si stamp prepared by UV lithography, kindly provided by Maya Gulic, from Experimental Physics Department of Ulm. The moulding process involves at rst the PDMS prepolymer mixing with a crosslinker in the ratio of 10:1 and degasication. This process occurs in a glass bell con-nected to a vacuum pump, and lasts 20 minutes. This is important to prevent air bubbles formation during the solidication procedure, since, especially at high temperatures, the trapped gas can expand and grow the bubble size.

Subsequently the mixture is cast on the Si mould as a homogeneous lm and cured in oven at 60°C overnight. The higher temperature activates the crosslinking process is accelerated and completed and the day after the PDMS is ready to detach from the master. In this case polymer stamps with holes of 2-3-4 and 5 μm diameter have been produced. As shown in Figure 2.8 their surface is reasonably homogeneous and the formed objects appear well dened.

10 μm 10 μm

(a) (b) (c) (d)

Figure 2.8: Optical microscope images (100X magnication) of PDMS stamps with circular holes of dierent diameters, of (a) 2 μm, (b) 3 μm, (c) 4 μm and (d) 5 μm. Production of ordered arrangements of polymeric spheres The substrate and the PDMS stamp are ready. The next step is a procedure to obtain ordered arrangement of the polymer spheres on the PMMA lm.

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The rst one, that is more conventional and mostly used in literature [25], requires the controlled distribution of the spheres in the stamp holes followed by a transfer process.

The second one, specically developed in this thesis, allows the correct par-ticles location directly on the substrate.

In both techniques the particle arrangement in the holes is driven by capillary forces. Their transfer is due to the dierent hydrophilicity of the stamp compared to the polymer layer on the glass support [25]. In particular, the polymer spheres are transfered from the more hydrophobic surface to the hydrophilic one. Figure 2.9 illustrates both procedures. The spheres only remain on the PMMA if its surface is properly hydrophilicized. The PDMS stamp is cut in squares of 5x5 mm2 _{and hydrophilicized with a plasma cleaning procedure during 1 minute [51].} For this process, a quantity of 10 μl of the already sonicated 3 μm size particles suspension is deposited on the PMMA layer with a micropipette.

(a) (b)

Figure 2.9: (a) The particles ordered on the stamp are transfered on the lm with a wet technique, (b) the empty master is applied on the spheres suspension on the lm.

Both simple casting and spin coating have been attempted, nding that the most signicant parameter is the polymer surface hydrophilization. Ordered areas of at least 30X30 μm2 _{were achievable, as depicted in Figure 2.10. At this point the} PMMA covered glass support is hydrophilicized with a plasma treatment of 20 minutes [51]. Immediately after this procedure, 5 μl of double distilled millipore-Q water is dropped on it and the PDMS stamp with the spheres located in the holes is superimposed on the wet surface. The samples were dryed at 25°C, overnight. Afterwards the stamp was removed and the polystyrene particles were transferred on the PMMA surface. The new procedure proposed in this thesis work consists in the direct application of the empty mould on the hydrophilized PMMA. Using a micropipette, 5 μl of polystyrene suspension was dropped on the polymer surface and then the PDMS stamp was applied on top of it. The specimens are dried with the same procedure of the previous method.

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10 μm

Figure 2.10: Optical microscope image (100X magnication) of Polystyrene parti-cles located in PDMS stamp holes.

As presented in Figure 2.11, both methods bring to a partial transfer of the polymer spheres from the mould (or the liquid) to the substrate. In the second case, the ordering process occurs during solvent evaporation, being still due to capillary forces. The fabrication method could handle also smaller particles, but it would require holes with smaller size in the polymeric stamp, not available during the thesis work but easily to realize.

10 μm 10 μm

(a) (b)

Figure 2.11: Optical microscope image (100X magnication) of (a) transfered patterns from the PDMS stamp, (b) ordered particles by empty mould superim-position.

Production of metal hemispherical cavities

Once that the polystyrene particles are correctly arranged on the substrate, the subsequent steps are: (i) the sputtering deposition of a thin metal lm and (ii) the superposition of another material to act as a mechanical stabilizer. Afterwards the initial glass substrate is detached, and the polymer spheres are dissolved by a suitable solvent.

The gold sputtering deposition The metal deposition is realized in cleanroom with the sputtering deposition apparatus Bal-Tec SCD 005 sputter coater, sketched in Figure 2.12.

A gold layer of 100 nm thickness has been obtained, in 280 sec, with a constant build-up rate of 0.357 nm/s. This rate is xed by the set current operation to 60 mA. This technique is quite fast and exible, inasmuch allows the employment of

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dierent metals. A schematic illustration of the technique is given in gure 2.12. The apparatus vacuum chamber consists in a glass cylinder with a low pressure level inside, (about 50 μbar), were an Ar gas is injected. The sample holder together with the lower metal support acts as a capacitor plate. The oscillating current determines electric eld oscillations between the dishes and this brings to the formation of a periodically moving plasma of charged Ar+ _ions.

vacuum pumps _{Ar injection} gold target

sample Ar+ plasma

vacuum chamber

A

Figure 2.12: Schematic illustration of gold sputtering deposition.

It happens that on both surfaces there is an ionic bombardment, which is mostly concentrated on the gold target. Sputtering of small material particles, tipically under the 10 nm size is produced, which will be deposited all over the exposed surfaces obtaining homogeneous deposited layers. Gold has been preferred to silver for the its better resistence to chemical degradation, very useful because of the chemical treatment to which the substrate is undergone. Figure 2.13 shows the results of the metallization on the samples.

10 μm

Figure 2.13: Optical microscope image (60X magnication) of gold covered polystyrene particles.

At this stage the polymer particles, covered of gold, already represent hemispheri-cal gold structures. Indeed, metal is only present on the upper side of the spheres,

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while the lower part is only partially covered. The degree of metal coverage of the polymeric spheres depends on the isotropicity of the metal deposition technique, in this case the sputtering technique [24]. In fact a perfectly isotropic deposition would lead to polymeric spheres almost completely covered by metal. In the case that the metal is deposited along a well collimated deposition direction from the top to the substrate, only the upper part of the spheres is metal covered.

The resin superposition and substrate detachment What is required now is a material for embedding the produced structures which can lead to more robust samples, allowing further physical and chemical treatments. Already available techniques are the utilization of an ahdesive tape or PDMS for embedding the metallized polymer particles [26, 44, 45]. In fact, depositing a PDMS layer or using the adhesive tape on the discussed paticles, it is possible to subsequently detach them from the glass substrate. The use of thermoplastic polymers, attempted by us without success in some preliminary tests, should be avoided, since they can be easily aected by swelling or even dissolution depending on the solvents used.

10 μm

Figure 2.14: Optical picture (60X magnication) of gold covered polystyrene par-ticles after the substrate detachment

The best method is to use a crosslinking epoxy resin: a mixture of epoxy resin (236390 Epoxydharz) and an ammine based crosslinker (Haerter L) with a weight ratio 10:3 has been prepared and degassed for 20 minutes in vacuum. Then the material is poured on the sample and a 24x24 mm2 _{glass substrate is superposed} on it. The samples are kept in oven at T = 60°C for 8 hours for a complete cross-linking and resin hardening.

Samples are subsequently soaked in double distilled millipore water overnight. During this process the PMMA layer deposited at the bottom swells, enabling the glass substrate detachment from the rest of the specimen. Turning it upside down by 180° the metal uncovered contact point of the polystyrene spheres with the

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PMMA becomes visible, as well as the gold layer deposited on the at area, as in Figure 2.14.

The PS spheres dissolution What is required in order to obtain empty metal cavities is to dissolve the polymer spheres located inside. One of the main prob-lems at this level is to distinguish any polymer residual inside the spherical holes using the optical microscope. AFM has been employed to obtain high resolution topographic images to clarify this issue ( for more details, see the chapter 4.10). Chemical procedures, as the employment of dierent solvents, , have been used to remove polystyrene [36]. The samples have been washed with acetone and toluene. They have been even sonicated in order to facilitate the uid access into the holes. After three washing cycles there is no more polymer left on the metal surfaces. Figure 2.15 shows the result.

10 μm

(a) (b)

Figure 2.15: Optical picture (100X magnication) of (a) partially cleaned and (b) empty cavities.

The insertion of gold nanoparticles

This last step was the production of a concentrated nanoparticles suspension and their insertion inside the gold cavities. The sizes of cavities and nanoparticles chosen for the preliminary trial of this thesis work are the consequence of a com-promise. The size of the cavity is limited, as explained before, from that of the available PDMS mould used to order the PS spheres. The gold nanoparticles must be small enough to provide decent SERS signal but not too much in order to avoid disordered clusters. In fact, the size of used gold nanoparticles was 400 nm, and particle accumulation after dipping the suspension on the cavities was obtained as detailed in the following.

Because of the fact that the cavities diameter is 3 μm and the employed gold nanoparticles 400 nm it was not easy to obtain holes with a single particle inside. The nal product shows the presence of more nanoparticles in each cavity. The

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usage of smaller nanoparticles leads to bigger aggregation problems and the con-sequent formation of particle aggregates on the surface. It would be possible, with smaller cavities with respect to the spheres, to adjust the relative sizes in order to achieve holes with a single particle inside.

The introduction of nanoparticles into the cavities Gold nanoparticles with diameter of 400 nm have been were purchased by Sigma-Aldrich, in water so-lution. Citrate is dissolved into the liquid as surfactant for particles agglomeration prevention. Before deposition of the spheres on the samples it is required to change the suspension solvent, from water to ethanol, because the relatively high water surface tension, 71.97 mN/m [46], may act as a limit for the objects penetration into the holes. The procedure, reported below, consists into three steps:

300 μl of nanoparticles suspension are taken with a pipette after sonication. Then the liquid is deposited in a small container and centrifugated for 5 min-utes at 13000 rpm. This procedure bring to the visible spheres accumulation at the bottom.

250 μl of solvent are removed with a pipette and 1200 μl of ethanol are added to the particles. The solution is sonicated in order to dissolve the aggregated gold nanoparticles and put them in contact with ethanol.

The specimen is centrifugated again and an amount of 1100 μl of solvent is extracted with the pipette. After one last sonication the concentrated ethanol-gold nanoparticles suspension is ready to be employed.

10 μm 10 μm

(a) (b)

Figure 2.16: Optical picture (100X magnication) of cavities with nanoparticles inside. (a) lower and (b) higher light focal plane.

The sample with the prepared gold cavities is soaked into ethanol during soni-cation. This procedure allows the solvent penetration inside the spherical holes.

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Immediately after this process 10 μl of concentrated nanoparticles suspension are deposited on the specimen surface with a micropipette. It is dried at 25 °C for 3 hours in a plastic box. During this step the ethanol evaporation on the at surface around the cavities moves the particles and determines the penetration of some of them inside the holes. Figure 2.16 shows some cavities with gold nanoparticles inside. Using the optical microscope is not possible to distinguish single particles, because of its resolution limit. In fact they look like granular structures, but thay can be resolved with the AFM.

Further improvements and developments of the fabrication method For more considerable plasmonic eects, as localized and delocalized plasmons, smaller cavities are required [31, 42]. For this reason one of the most reasonable improvement of this preparation method would be the reduction of holes dimension down to 0.5 μm. Employment of smaller PS particles would be necessary to this purpose. Even if multilayers formation could occur during the self-assembling process, this drawback can be overcome by suitably choosing the properties of colloidal suspension. The PDMS stamp production with holes even of the size of 100-200 nm would be achieved without diculty with the same technique involving UV lithography and Si moulding. The transfer technique would work even in this smaller range, as already demonstrated [25]. There is no specic reason why the subsequent steps should not equally work in this context.

Another technical interesting development would be the use of a soluble substrate where to obtain an ordered arrangement of particles. Instead of using glass with a PMMA layer and then detach it from the rest of the specimen after the resin application, in this case the employment of a simple solvent could remove it. Even PMMA or PS would be used as a substrate for this issue. In order to obtain a regular and at enough polymeric surface it would be sucient to deposit some liquid solution of the material on a glass and then separate it after drying. The side on contact with the substrate would be ready for the spheres arrangement. With this system the polymer spin coating, the soaking into water and detachment steps would be avoided, leading to a considerable time saving, since the bottom substrate and the PS particles would be dissolved in only one single step. Furthermore, the prevention of the mechanical glass separation would lead to the fabrication of more complete cavities than hemispherical [26]. In eect the thin metal layer on top of the cavities may be broken and removed together with the substrate during this process, especially in the case of below micron sized holes.

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almost comparable size. Metal spheres under the size of 500 nm are commercially available. In this case cavities with only one particle inside would be obtainable. This system would be simpler to simulate and predict in terms of plasmonic eects [34]. Depending on the relative sizes of two objects even structures with two spheres inside (dimer) may be designed and realized, such as those shown in Figure 2.17:

(a) (b)

Figure 2.17: (a) single gold nanoparticle, (b) dimer in a cavity.

Futhermore, the gold thickness of the cavity is controlled by the amount of de-posited metal. This means that the proposed technique, unlike the usual electro-chemical deposition techniques [61], allows the control of the electrons connement in the direction of the material thickness, with important consequences on the LSPR tuning. This would combine the plasmonic local eld enhancement, typi-cal of nanoparticles, dimer or trimer structures, to the already present plasmonic eects and light trapping due to the spherical metal cavity [34, 38, 35].

2.2 Experimental details of the characterization

tech-niques

In order to analyse the fabricated micro and nano-structures both the atomic force microscope (AFM) and the confocal Raman microscope (CRM) have been employed. In the following sections these two instruments are described. The rst technique will be just briey described, since nowadays it is a well known technique, while the second one is introduced in more detail. The AFM has been used in order to obtain a morphological characterization of the metal structures. In fact these measurements allow the evaluation of structure size and shape, with nanometer scale resolution. Using the CRM it is possible to study the optical properties of the substrates, both in terms of elastic and anelastic Raman scattering.

2.2.1 The atomic force microscopy (AFM)

The atomic force microscopy (AFM) is a valuable technique for the analysis of nanostructured substrates. It allows high spatial resolutions, due to the local

Fabrication and characterization of micro and nano-structures for surface-enhanced Raman spectroscopy

Contents

Introduction

Thesis structure

Chapter 1

The physics of the surface-enhanced

Raman scattering

1.1 The Raman scattering

1.2 The resonant Raman scattering

1.3 The surface-enhanced Raman scattering (SERS)

1.3.1 The chemical enhancement

1.3.2 The electromagnetic enhancement

1.3.3 The electromagnetic theory of SERS

1.3.4 The Enhancement Factor (EF) calculation

Chapter 2

Experimental techniques of

fabrication and characterization of

SERS structures

2.1 Sample micro-nanofabrication

2.1.1 The nanosphere lithography

10 μm

10 μm

(b)

(a)

(d)

(c)

2.1.2 Fabrication of microcavity localized nanoparticles

2.2 Experimental details of the characterization

tech-niques

2.2.1 The atomic force microscopy (AFM)