Studies of the interface between molecular films and solid supports are in focus point of my research. Determination of changes in the orientation, structure and reactivity of molecules present in various supramolecular assemblies at solid surfaces, due to reactions caused by external impulses such as binding agents, electric potentials or temperature are of large research interest to me. An ideal analytical method is one which would allow for carrying out sensitive, selective, reproducible, fast, and in situ chemical measurements of the composition and function of functional molecular systems. Therefore, in my research advanced structure characterizing techniques are used for in situ studies of various molecular assemblies.
A biological cell membrane is the most important charged interface in nature. Assuming a membrane thickness of 6 nm and potential drop in the range of 90 to 200 mV (Figure 1A), electric fields acting on lipids and proteins in the membrane are in order of 1 ×107 – 3 ×107 V m-1. Under these electric fields the structure, orientation as well as the reactivity of a molecule may differ from properties measured in a bulk phase. Thus, it is of high priority to detect in situ intermolecular interactions and describe them to structural changes under controlled electric fields.
Physical chemistry possesses analytical methods suitable to approach this challenging task. Deposition of molecular films such as lipid bilayers on an electrode surface ensures their exposition to varying electrical fields (Figure 1). Electrochemical methods measure integral macroscopic properties of studied supramolecular assemblies such as the capacitance, charge, potential drop, permeability or coverage. However, they are not sensitive to the structure of molecules adsorbed on the electrode surface. Determination of the structure of molecules adsorbed on electrode surfaces requires use of spectroscopic and/or microscopic methods which may be combined to electrochemical techniques.
Infrared spectroscopy (IRS) belongs to one of the most powerful techniques for the analysis of the composition and structure of organic molecules. Moreover, it gives the unique opportunity to analyze simultaneously each kind of species present in a sample. In contrast to other spectroscopic techniques, a complex composition of the sample limits neither the spectroscopic resolution nor sensitivity. Since various functional groups present at various molecules absorb the infrared light at specific frequencies, the influence of each component on the structure of the entire assembly can be studied simultaneously without labeling with any molecular probes. In addition, IRS is applicable not only to study the molecular structure in a bulk phase but also in molecular assemblies present at various interfaces. Infrared reflection absorption spectroscopy (IRRAS) is a non-invasive excellent tool for analyzing the physical state, structure, and orientation of a molecule adsorbed on a reflecting substrate. Particularly attractive is polarization modulation infrared reflection-absorption spectroscopy (PM IRRAS), due to reduction of the background contribution.
1. Spectroelectrochemical studies of redox-inactive films adsorbed on electrode surfaces
Combination of electrochemistry to PM IRRAS results in an absorption spectrum of an adsorbed molecule recorded at a selected potential applied to the electrode surface. A set of PM IRRA spectra in the CH stretching modes region of the hydrocarbon chains in a phospholipid bilayer adsorbed on the gold electrode surface as a function of potential applied to the gold electrode is shown in figure 2. The potential scan reflects the adsorption – desorption process of the lipid bilayer on the Au electrode surface (more 1|2|3)
Figure 2 shows large changes in the intensities of the methyl and methylene stretching modes in a lipid bilayer adsorbed on the gold surface. They reflect changes in the orientation of the molecules adsorbed on the electrode surface. The quantitative analysis of PM IRRA spectra, measured at different potentials, provides information on the structure, orientation, hydration and packing changes of molecules present in the film adsorbed on the electrode surface. From these analyses a sub-molecular scale picture of a supramolecular assembly is available.
Lipid membranes interact constantly with proteins. In situ PM IRRAS is an excellent analytical tool to study lipid-protein interactions. The neuronal calcium sensor protein, recoverin, is expressed in retinal rod and cone cells and is involved in the calcium-dependent control of receptor (rhodopsin) phosphorylation and receptor inactivation. In its Ca2+-saturated form recoverin is attached to membranes by an exposed myristoyl acyl chain. Figure 3 show PM IRRA spectra of a lipid bilayer alone and upon its interaction with recoverin.
The presence on the amide I mode proves that recoverin is attached to the lipid bilayer. The number of contributions to the amide I and their frequencies reflect the secondary structure of proteins. Deconvolution of the amide I mode from proteins adsorbed in a solid surface or attached to a lipid bilayer allows for the determination of the secondary structure elements in the studied protein (more 1|2)
Electric potentials affect the structure of biopolymers (e.g. proteins or DNA) adsorbed on electrode surfaces. Obviously, PM IRRAS with electrochemical control is the perfect method to investigate structural changes in these assemblies (more 1|2)
Recently, we used in situ PM IRRAS to follow potential depended changes in the orientation of base pairs in DNA fragments adsorbed on the gold surface. Figure 4 shows the PM IRRA spectra on the C=O and ring stretching modes region of an adenine-thymine (dAdT)25 fragment.
Excellent signal-to-noise ratio of the measured in situ PM IRRA spectra allowed us to deconvolute this busy spectral region, make band assignment and perform orientation analysis of two complementary bases with respect to the Au electrode surface (more). At positive potentials tilting of the long axis of the DNA helix is observed. Detailed spectral analyses show that at negative potentials the helices are vertically oriented with respect to the Au surface. However, the relative orientation of the complementary bases changes, providing clear evidence that electric potentials contribute to the stability and hybridization of the DNA double helix.
2. Spectroelectrochemical studies of redox-active films adsorbed on electrode surfaces
Electron transfer reactions may influence the structure and orientation of redox-active molecules present in films on electrode surface. Currently we investigate the impact of electron transfer reactions on the structure and orientation of redox active metalloorganic surfactants in Langmuir-Blodgett films and in coordination network compounds deposited on the gold electrode surface.
Transition metal hexacyanometallates represent the oldest known group of coordination network compounds that has been investigated for many years due to their interesting sorption, magnetic, electrochromic, electrocatalytic or sensing properties. While those electrode modifications are easily accessible from a preparative point of view, the detailed assignment of the observed voltammetric signals to particular redox transitions and elucidation of consequential structural changes in those materials has remained controversial. The n(CºN) is an excellent indicator for the determination of the composition, oxidation state and binding motifs in metal hexacyanoferrates. Figure 4 shows PM IRRA spectra of cobalt hexacyanoferrate film on gold.
Figure 5 clearly shows that the number, frequency and intensity of the n(CºN) modes depend strongly on the potential applied to the gold electrode. A blue shift of the n(CºN) stretching mode is observed with an increase in the oxidation state of the metal ion. This experiment allows us to determine the pathway of redox reactions taking place in the cobalt hexacyanoferrate film. Briefly, the redox reaction at E1°’ = 0.53 V involves Co(II/III) centers while the electron transfer at E2°’ = 0.685 V occurs on the Fe(II/III) sites.
3. Gold is the perfect substrate for in situ PM IRRAS. Are any other materials also attractive?
Application of in situ PM IRRAS is limited to surfaces which reflect strongly the IR light and are conductive. In order to increase applicability of this superb surface analyzing technique new materials are tested. We have shown that hybrid titanium oxide carbide is an interesting material for application in PM IRRAS (more). This material reflects strongly the IR light and conducts electric current. Figure 5 shows the PM IRRA spectra of arachidic acid monolayer adsorbed on the titanium oxide carbide (upper panel) and gold (lower panel) substrates.
Clearly, this hybrid material is suitable for in situ PM IRRAS investigation of monomolecular films adsorbed on their surfaces.
Recently we have followed potential dependent structural changes in redox-active polymer films on a glassy carbon electrode (GCE) surface. As a non-metallic surface, GCE provides less favorable conditions than classical PM IRRAS substrates such as gold. However, a careful optimization of the optical configuration allowed the use of GCE for polymer films that considerable exceed the thickness of monolayers.
A redox-active plumbagin polymer film shows complex electrochemical characteristics. Parallel oxidation reactions occur in the polymer films at similar potentials. The progress of redox reactions in this polymer film depends on the experimental conditions such as use of H2O and D2O and the pH (pD) of the electrolyte solution. PM IRRAS with electrochemical control was employed to follow potential-dependent structural changes in the polymer film (more). Figure 7 shows a photograph of the spectroelectrochemical cell with the glassy carbon electrode covered by plumbagin polymer film.
Currently realized research projects:
-Spectroelectrochemical studies of the impact of the lipid–protein interaction on the structure of a model lipid bilayer exposed to physiological electric fields.
-Fabrication of realistic asymmetric models of bacterial cell membranes. Studies of their interactions with antibacterial peptides.
-Spectroelectrochemical studies of potential-dependent changes in the orientation and hybridization of DNA fragments adsorbed on electrode surfaces.
-Studies of structural changes induced by electron transfer reaction in organized films of redox-active metallosurfactants in thin organized films for application as electronic devices.
-Spectroelectrochemical studies of orientation of charged amphiphilic molecules in Langmuir-Blodgett films as models of electrochemical double layer in ionic liquids
-In situ spectroscopic studies of the protein adsorption on solid surfaces. Application of IRS for studies for in situ determination of structural changes in proteins aiming at understanding of the protein folding and misfolding processes.
-Development and test of new, conductive, non-metallic surfaces for in situ spectroelectrochemical experiments.
-Determination of structural changes accompanying corrosion process of light metals: a combined electrochemical, PM IRRAS and XPS study.