As it was previously discussed, layer-functionalized surfaces present novel structural properties that influence their response to external stimuli. In order to interpret correctly their behavior and modulate it for the envisaged applications, an investigation technique should ideally be able to correlate the surfaces composition (in terms of arrangement on the substrate, intermolecular orientation and packing) with their reactivity in real working conditions (i.e. in situ or operando). For this purpose, several methods have been developed throughout the years, aiming more and more to increase the analytical sensitivity from macro to micro and, most recently, even to nano structures. In the following paragraphs we will briefly review a few of the more striking analytical techniques allowing to characterize in situ or operando the properties of molecular layers, thiol- and diazonium-based layers mostly.
Quartz Crystal Microbalance
The working principle of QCM relies on the use of a disk made of a piezoelectric material, i.e. of crystalline structures with no inversion symmetry, which can deform mechanically under the application of an external electric field across two thin metal electrodes deposited on each side. Vice versa, these materials can be electrically polarized when subjected to a mechanical stress 73. In both cases, the intensity of the deformation and the polarization are proportional to each other. Hence, an alternate voltage (MHz regime) will induce a constant oscillation on the crystal and can excite the crystal to resonance, the frequency of which depends on the thickness (mass) of the disc. When the piezo-material is modified by the deposition of a molecular layer on its surface, a shift in the resonance frequency is recorded and can be related to the mass of the deposited layer thanks to the Sauerbrey relationship. The sensitivity of the technique, called microbalance, can detected mass variations (increase, decrease) ranging from several hundreds of micrograms down to nanograms .
Raman effect: an inefficient inelastic scattering process
It is also possible that the photon energy is intermediate between the ground level and the first excited electronic level; in this case, absorption cannot occur and the photon will be scattered. Most commonly, the scattering occurs elastically, hence the scattered photon maintains the same energy as the incoming one (Rayleigh scattering). However, if the photon is scattered inelastically, its final energy will show a negative (Stokes shift) or positive (anti-Stokes shift) variation that matches the energy between two vibrational levels. While the signal originated by the elastic scattering does not gather any chemical information on the sample, the inelastic scattering is at the basis of the Raman technique 85.
Despite the intrinsically different nature of the Raman scattering with respect to the IR absorption, both techniques are in fact employed to probe the transitions among vibrational levels in the studied systems and can sometimes yield complementary information. This is due to their particular “selection rules”, i.e. to the conditions a vibrational transition should fulfil to be detected by either of the two spectroscopic techniques. Specifically, absorption in IR will be observed if the transition generates a variation in the molecular dipolar moment, while Raman scattering will be produced when a change in the molecular polarizability occurs. Because vibrations often modify only one of the two parameters and leave the other unaffected, IR and Raman results are usually considered as complementary 86.
As in this work we mainly employed Raman spectroscopy, we will focus in the following on its description. Raman scattering experiments are generally easy and straightforward to perform, as they do not require any sample preparation and can be performed in normal ambient conditions. In a basic setup, the sample is mounted on the sample stage and illuminated through a confocal microscope (see Chapter 2) with a visible or NIR laser light source. The signal scattered back from the sample is collected again by the objective and directed towards the spectrometer, where it is detected. According to Equation (1.1), the achievable lateral resolution reaches values down to a few microns. Note that the spatial resolution in Raman spectroscopy is nearly constant along the spectrum, as a typical Raman shift (up to 4000 cm-1) is contained within 30 to 200 nm depending on the monochromatic excitation wavelength. This is not the case for IR spectroscopy, which uses broad band sources like globars. The range of the mid-infrared domain is typically between 4000 cm-1 and 400 cm-1 (or between 2.5 μm and 25 μm)., thus the spatial resolution varies along the spectrum between ~1 to 10 μm. This aspect, along with the lower sensitivity of Raman spectroscopy to water (whose vibrations are accompanied by small changes in the polarizability, hence by weak scattering), makes it preferable to the IR technique, especially in those applications (such as biological and medical compositional analyses 87–89) where the samples need to be kept in a wet state.
However, standard Raman analyses on nanostructured materials are not expected to detect precisely the characteristics of single adsorbed molecules, but rather to obtain a collective signal averaged over large (micrometric) areas. Besides, because of the low Raman scattering efficiency, signals are usually very weak and difficult to detect when arising from thin molecular layers. In general, when a molecule scatters an external light source, the total intensity of the radiation is proportional to the square of the induced dipole moment: 𝐼=16𝜋43𝑐3𝜈4∙𝜇𝑖,02 (1.3).
where 𝑐 is the speed of light and 𝜇𝑖,0 is the amplitude of the induced dipole moment. Due to its dependence on the fourth power of the frequency 𝜈, the scattered intensity is much stronger for radiations of short wavelength, i.e. high frequency. The intensities of the scattered lines depend also on the intrinsic properties of the studied system, more specifically on the cross-section 𝜎, which is defined as the ratio between the total scattered power (in W) and the irradiance of the incoming radiation (in W.m-2) and has therefore the dimensions of an area. For a single Stokes transition i, the number 𝑁𝑠 of light quanta inelastically scattered per atom, per length of material 𝑑𝑧 per solid angle element 𝑑𝛺, can be defined as 90: 𝑑𝑁𝑠=𝑁𝑎𝑁𝑜(𝜕𝜎𝑖𝜕𝛺)𝑖𝑑𝛺𝑑𝑧 (1.4).
where 𝑁𝑎 is the number of molecule per unit volume in the lower energy state, 𝑁0 is the number of incident light quanta and the ratio (𝜕𝜎𝑖𝜕𝛺)𝑖 is the differential cross section, described by a complex formula 91. Respect to the other optical spectroscopies, the Raman effect appears to have the lowest cross section: averagely, for an incident flux of 108 photons, only one photon will be inelastically scattered. For comparison, a single fluorescent impurity with a quantum yield of 0.1 can produce ten fluorescence photon for the same incident flux. In summary, not only the Raman transitions are difficult to observe, but also they can be easily and completely covered by other light-emitting effects (as fluorescence).
Scanning electrochemical cell microscopy
Scanning electrochemical cell microscopy (SECCM) can be considered as an evolution of SECM, implemented with the aim of reaching single-entity sensitivities. Instead of an UME, in SECCM the probe is constituted by a single or double barrel (theta) nano- or micropipette, filled with the electrolyte solution and integrating a quasi-reference electrode (QRE). Similarly to what was observed for Raman-μSEC, also in this case the interactions between the probe and the substrate are limited by a meniscus of few micro- or nano-liters, which localizes the origin of the signal from a restricted sample area. The pipette can therefore be scanned over the surface and collect an array of measurements, which will yield a surface reactivity mapping.
For instance, SECCM was shown to reach better resolution than conventional SECM in the detection of pinholes in a 4-NBD-derived layers grown onto glassy carbon: thanks to the coupling with numerical simulations, details down to 20 nm, much smaller than the pipette size itself, could be successfully resolved 138 (Figure 1.23).
Electrochemical tip-enhanced Raman spectroscopy
In the panorama of nanoscale dynamic investigation techniques, electrochemical tip-enhanced Raman spectroscopy (EC-TERS) can be considered as a very successful hyphenation of powerful and complementary techniques. Indeed, EC-TERS gathers together the atomic/molecular-size spatial resolution provided by scanning probe microscopy (as STM or AFM), the extraordinary chemical sensitivity and wealth of information of SERS (chemical signal enhancement) and even the characteristics of an electrochemical probe, which can be conveniently employed for the investigation and/or promotion of electrochemical reaction under in situ and operando conditions. In the next paragraphs we will first introduce the original developments of the TERS technique and its implementation within different SPM setups. Later on, we will focus on the most recent state-of-art configurations that allows its use in liquid and under electrochemical conditions. Finally, we will present the issues and challenges that are currently impeding the large and fast development of this technique.
First TERS developments
A first precursor of the TERS technique can be considered the scanning near-field microscope (SNOM). The principle of this technique was first proposed theoretically by Synge in 1928 as a solution to overcome the problem of diffraction-limited spatial resolution in optical microscopes 156. Synge’s idea consisted in illuminating the sample through a minute aperture, whose diameter is smaller than the wavelength 𝜆 of the beam that crosses it, and that is placed at a distance z (≪𝜆) from the measured surface, i.e. at near-field distance 84,156. Thanks to this expedient, the lateral resolution was expected to be no longer limited by diffraction (i.e. by the use of optic lenses), and to depend only on the size of the aperture (which determines also the sampled volume) 84. Almost 60 years passed before Synge’s idea could be put in practice: the breakthrough consisted in the invention of STM and, successively, of the other SPM techniques that allow placing a probe at very close proximity to the sample surface 84.
In order to improve the resolution and, at the same time, intensify the signal in SNOM, it was proposed to substitute open optical fibers with apertureless probes, whose tapered apexes can enhance the electromagnetic field of the far-field illuminating source 157 and behave like a light scatterer. This had first been theorized when it was observed that the light travelling inside an open probe had a maximum enhancement in the lobes near the aperture rims. This means that field confinement was limited by the size of the aperture diameter, which should have been reduced to a single point to provide the strongest field localization (and corresponding enhancement) 157, as it was also observed for the tip-SERS technique. Moreover, because of the lateral resolution dependency on the tip diameter, reducing this parameter was also expected to improve the imaging quality.
TERS in liquid and under electrochemical conditions
A much ambitious task endeavored this last decade in the TERS community has consisted in implementing the TERS setups under liquid conditions. Liquid-TERS indeed allows to perform in situ imaging of either more sensitive samples, as biological tissues, which need to be kept in wet state in order not to collapse 3, or of functional devices under their conditions of operations (operando analyses). A first success was achieved in 2009 by Zenobi’s group, which carried out measurements over a thiophenolate (PhS) SAM on gold in liquid environment thanks to an Ag-coated tip, protected with an ethanethiolate (EtS) layer and mounted on an inverted AFM setup 3. As Figure 1.31a shows, the measurements were performed in the solution meniscus between the tip scanner and the sample. Besides, the tip movements could be precisely followed thanks to presence of a window just above the cantilever head that allowed focusing the tracing laser beam; this did not interfere with the laser beam used for TERS measurements, since the system worked in bottom-illumination mode and back scattering 3. The presence of the EtS protective layer was shown to be essential to avoid the contamination of the tip apex with the thiol sample, which could partially re-dissolute and adsorb on the silver surface. Although the presence of this coating affects the EF (which decreases from 104 to 103), the obtained signal was still very sharp and resolved (Figure 1.31b).
SPM/ Raman coupling platform and choice of the illumination geometry
The OmegaScope 1000 used in this work and presented earlier on Figure 2.5 was specifically designed to optically couple Raman spectroscopy with the SmartSPM microscope. The latter is provided with different measuring heads depending on the imaging mode implemented, i.e. AFM, shear-force microscopy (ShFM, using tuning forks) and STM. Only this latter was used in this work, in order to carry out ex or in situ STM-TERS experiments.
The STM head consists in a simple platform holding in place both the tip holder and a conductive unit connected to the tip and the sample (Figure 2.7a.). The conductive unit establishes a bias voltage between the tip and the sample (typically 0.1 V) and also measures the net tunneling current flowing between them. Several amplifications gains are available, allowing currents from pA to μA to be measured. The current feedback loop should have a bandwidth large enough to allow fast adjustment of the tip-sample distance. However, since the noise level increases with the bandwidth, this should also not be too large. In this work, low-pas cutting frequency was set to 25 kHz.
The open design of the microscope head (clearly visible in Figure 2.7b) allows a high flexibility over the experimental conditions, easing, for instance, the introduction of more elaborated tip and/or sample holder designs or the coupling to external instrumentations (see EC-STM-TERS setup, presented later). Note that on the SmartSPM microscope, a sample piezo-scanner ensures the raster scanning for topography/composition imaging of the sample without compromising the optical coupling, i.e. the precise focusing of the laser beam on the tip apex using a piezo-controlled objective lens (carried by the OmegaScope coupling platform). This represents an advantage respect to Ren’s STM-TERS configuration, described in Section 1.3.3. The accessible x, y, z scanning range of the sample scanner is 100x100x15 μm, 30x30x10 μm for the objective scanner.
First prototype of the EC-STM-TERS setup
When performing EC-STM-TERS, a 4-electrode system must be used, where the tip and the sample act both as working electrodes (WEs), whose potential is set against a reference electrode (RE), while the counter-electrode (CE) provides the required current flowing through the two WEs. This double potential regulation implies the use of a bi-potentiostat, the major difficulty here being to integrate to it the conductive unit of the SmartSPMTM. This is necessary to ensure the efficient control over the STM tip-sample distance using the tunneling current as a feedback parameter. A dedicated bi-potentiostat was designed in LISE to provide potential control and exploration while maintaining a constant bias voltage between the tip and the sample, and to enable the proper amplification of the tunneling current measurement by the conductive unit.
Figure 2.9 shows the cell prototype designed by T. Touzalin 125. The sample is immobilized on a poly-ether-ether-ketone (PEEK) support, and above it is glued to a rubber O-ring, which defines the actual volume of the electrochemical cell (capacity of ~1 mL of electrolyte) and constitutes its wall. Both the rubber and the PEEK are resistant to aqueous and organic solvents. A gold wire, following the contour of the O-ring inner walls, is used as CE, while a silver wire protruding inside the cell works as RE. Both are fixed to the PEEK support and, along with the gold surface, are connected to external bi-potentiostat by long and thin copper wires. The overall setup is quite bulky and cluttered, but sufficiently light and stable at the same time to ensure proper topography tracking by STM. However, within this design the electrodes are all very close to each other and their position is difficult to adjust, hence shortcuts can occur quite easily. Moreover, sample damage and contamination can easily occur upon gluing of the O-ring. Also, the thin and long wires employed as connectors for the electrodes are not shielded, with the risk of electrical noise and shortcuts.
Table of contents :
Chapter 1 State of the art
1.2. Surface modification
1.2.2 Diazonium salts
1.2. Dynamic investigations
1.2.2 Characterization at the macro/microscale
1.2.3 Characterization at the nanoscale
1.3. Electrochemical tip-enhanced Raman spectroscopy
1.3.1 First TERS developments
1.3.2 TERS developments in air
1.3.3 TERS in liquid and under electrochemical conditions
Chapter 2 STM-TERS under electrochemical conditions – Experimental developments & implementation
2.2. Elaboration of TERS-active bulk metal probes
2.2.1 Design of plasmonic amplifiers
2.2.2 TERS probe from etched gold wires
2.3. Raman and STM coupling: technical implementation
2.3.1 Optical construction of the Raman microscope
2.3.2 SPM/ Raman coupling platform and choice of the illumination geometry ..
2.4. EC-STM-TERS analyses: setups and modus operandi
2.4.1 First prototype of the EC-STM-TERS setup
2.4.2 Optimization of the EC-STM-TERS setup
2.4.3 EC-STM-TERS analyses – Step-by-step implementation
2.4.4 Assessment of the TERS signal stability with the potential
Chapter 3 Study of a complex reduction mechanism on a model sample
3.2. 4-NBM as model system
3.2.1 SAM sample preparation
3.2.2 Electrochemical measurements
3.3. EC-STM-TERS: real-time following of the reduction mechanism
3.3.1 EC-TERS experimental conditions
3.3.2 Mapping at fixed potential
3.3.3 Deciphering of the reaction mechanism by real-time TERS
3.4. Discussions and conclusions
Chapter 4 Diazonium-based structures: influence of the thickness on the electrochemical reactivity
4.2. Elaboration of functionalized surfaces by controlling diazonium chemistry
4.2.1 Electrochemical grafting of 4-NB radicals on gold
4.2.2 Control of 4-NBD layer growth with a redox cross-inhibitor
4.2.3 Characterization of the grafted layers
4.2.4 STM-TERS mapping in air
4.3. Surface reactivity screening by EC-TERS mapping
4.3.1 Measurement setup
4.3.2 TERS mapping under polarization
4.3.3 Structure-reactivity relationships
Conclusions and perspectives
Comparison of ChA and DPPH performances as inhibitors