Rheological characterisation of alginate-like exopolymer gels crosslinked with calcium.

Bacterial alginate-like exopolymers (ALE) gels have been used in this work as a model for the extracellular polymeric matrix of biofilms. Aim was to relate the mechanical properties and strength of this matrix that make biofilms as persistent to cleaning as they are, to the complex cohesive molecular interactions involved. Mechanical properties of the gels as a function of CaCO3 concentration were investigated using dynamic and static rheology. Gels with relatively low CaCO3 concentrations, between 100 µmol and 300 µmol per g ALE, were found to exhibit similar viscoelastic behaviour as real biofilms, with elastic moduli between 50 Pa and 100 Pa and dissipation factors between 0.2 and 0.3. Increasing CaCO3 concentrations resulted in an increase of the elastic modulus up to 250 Pa, accompanied by an increase in brittleness. At a CaCO3 concentration of 1250 µmol per g ALE this trend stopped, probably due to disturbance of the continuous ALE network by precipitation of salts. Therefore, overdosing of Ca salts can be an adequate approach for the removal of biofouling. All gels exhibited permanent strain hardening under medium strain, and their mechanical properties showed dependency on their strain history. Even after application of an oscillatory strain with 200% amplitude that caused the gel structure to collapse, the gels recovered 65 to 90% of their original shear modulus, for the major part within the first 20 s. Recovery was slightly less for gels with high CaCO3 concentration. In creep tests fitted with a Burgers model with multiple Kelvin elements at least three different interactions in the ALE gels could be distinguished with characteristic retardation times in the range of 10, 100 and 1000 s. Further identification of the mechanisms underlying the gel mechanics will allow the development of targeted strategies to undermine the mechanical strength of biofouling and aid the cleaning process.

Self-consistent field modeling of mesomorphic phase changes of monoolein and phospholipids in response to additives.

Mapping the topological phase behaviour of lipids in aqueous solution is time consuming and finding the ideal lipid system for a desired application is often a matter of trial and error. Modelling techniques that can accurately predict the mesomorphic phase behaviour of lipid systems are therefore of paramount importance. Here, the self-consistent field theory of Scheutjens and Fleer (SF-SCF) in which a lattice refinement has been implemented, is used to scrutinize how various additives modify the self-assembled phase behaviour of monoolein (MO) and 1,2-dioleoyl-phosphatidylcholine (DOPC) lipids in water. The mesomorphic behaviour is inferred from trends in the mechanical properties of equilibrium lipid bilayers with increasing additive content. More specifically, we focus on the Helfrich parameters, that is, the mean and Gaussian bending rigidities (κ and [small kappa, Greek, macron], respectively) supplemented with the spontaneous curvature of the monolayer (Jm0). We use previously established interaction parameters that position the unperturbed DOPC system in the lamellar Lα phase ([small kappa, Greek, macron] < 0, κ > 0 and Jm0 ≈ 0). Similar interaction parameters position the MO system firmly in a bicontinuous cubic phase ([small kappa, Greek, macron] > 0). In line with experimental data, a mixture of MO and DOPC tends to be in one of these two phases, depending on the mixing ratio. Moreover we find good correlations between predicted trends and experimental data concerning the phase changes of MO in response to a wide range of additives. These correlations give credibility to the use of SF-SCF modelling as a valuable tool to quickly explore the mesomorphic phase space of (phospho)lipid bilayer systems including additives.

Electrode Surface Potential-Driven Protein Adsorption and Desorption through Modulation of Electrostatic, van der Waals, and Hydration Interactions.

When proteins in aqueous solutions are exposed to solid substrates, they adsorb due to the dynamic interplay of electrostatic, van der Waals, and hydration interactions and do so in a rather irreversible fashion, which makes protein recovery troublesome. Here, we use a gold electrode as the solid substrate and modulate the surface potential to systematically induce protein adsorption as well as partial desorption. We use different methods such as surface plasmon resonance, atomic force microscopy, and electrowetting and show that biasing the electrode to more negative potentials (by -0.4 V compared to the open-circuit potential at pH 6) results in an increased adsorption barrier of 6 kJ mol-1 for the negatively charged protein ß-lactoglobulin. Further, we clearly demonstrate that this is due to an increased double layer potential of -0.06 V and an increase in hydration repulsion. This indicates that an electric potential can directly influence surface interactions and thus induce partial ß-lactoglobulin desorption. These observations can be the basis for biosensors as well as separation technologies that use only one trigger to steer protein ad- and desorption, which is low in energy requirement and does not generate large waste streams, as is the case for standard protein separation technologies.

Structural and mechanical parameters of lipid bilayer membranes using a lattice refined self-consistent field theory.

The self-consistent field theory of Scheutjens and Fleer is implemented on a grid with (lattice) sites that are smaller than the segment size. In this quasi lattice-free implementation we consider united atom-like molecular models and study bilayer self-assembly of phospholipids in a selective solvent (water). We find structural as well as mechanical parameters for these bilayers. The mean (κ) and Gaussian ([small kappa, Greek, macron]) bending moduli, as well as the spontaneous curvature of the monolayer (Jm0), are computed for the first time following a grand canonical ensemble route. Results are in line with previous estimates for mechanical parameters that at the time could not be made following this correct route. This proves that the mean bending modulus is only a very weak function of the membrane tension. We performed a systematic study on the effects of model parameter variations. The mean bending modulus generally grows with increasing bilayer thickness. As expected Jm0 and [small kappa, Greek, macron] behave oppositely with respect to each other and for classical phospholipids assumes values near zero. As an example, an increase in the lipophilic to hydrophilic ratio in the lipids, may cause the Gaussian bending rigidity to switch sign from negative to positive, while - not necessarily at the same point - the spontaneous curvature of the monolayer may switch sign from positive to negative. Together with other investigated trends, these results point to mechanisms of how topological phase transitions of the lipid bilayer membranes may be regulated in the biological context, which correlates with known lipid phase behaviour.

Step-wise linking of vesicles by combining reversible and irreversible linkers - towards total control on vesicle aggregate sizes.

Small vesicle aggregates as a model for primitive cellular assemblies or for application as multi-compartment drug delivery systems recently received a lot of interest, yet controlling the aggregation of vesicles to predetermined aggregate sizes remains quite a challenge. We show that this type of control is possible by using a combination of two different linker systems: streptavidin-biotin and C18-pNIPAm. The latter linker is a thermoresponsive surfactant, which below its lower critical solution temperature (LCST) of 32 °C acts as barrier on the outside of the vesicles preventing aggregation, even in the presence of other linkers. Above the LCST however, C18-pNIPAm collapses, becomes sticky and thus acts as a linker inducing aggregation. By working at low vesicle concentrations and tuning the C18-pNIPAm/lipid ratio, the aggregation is by design limited. When the temperature drops below the LCST again, the aggregation is reversed. However, this is not the case if other linkers are present. The collapse of C18-pNIPAm above the LCST provides close contact between vesicles, allowing other linker molecules to connect them. By combining the reversible 'switch-like' aggregation properties of C18-pNIPAm, with the irreversible linkage between biotinylated lipids and streptavidin, it is possible to control the size of the aggregates step by step using a simple temperature program.

Self-limiting aggregation of phospholipid vesicles.

Lipid vesicles are widely used as model systems to study biological membranes. The self-assembly of such vesicles into vesicle pairs provides further opportunity to study interactions between membranes. However, formation of vesicle pairs, while subsequently keeping their colloidal stability intact, is challenging. Here, we report on three strategies that lead to stable finite-sized aggregates of phospholipid vesicles: (i) vesicles containing biotinylated lipids are coupled together with streptavidin, (ii) bridging attraction is exploited by adding cationic polymers (polylysine) to negatively charged vesicles, and (iii) temperature as a control parameter is used for the aggregation of vesicles mixed with a thermo-sensitive surfactant. While each strategy has its own advantages and disadvantages for vesicle pair formation, the latter strategy additionally shows reversible limited aggregation: above the LCST of pNIPAm, vesicle pairs are formed, while below the LCST, single vesicles prevail. Mixing protocols were assessed by dynamic and static light scattering as well as fluorescence correlation spectroscopy to determine under which conditions vesicle pairs dominate the aggregate size distribution. We have strong indications that without subsequent perturbation, the individual vesicles remain intact and no fusion or leakage between vesicles occurs after vesicle pairs have formed.

A Hybrid Monte Carlo Self-Consistent Field Model of Physical Gels of Telechelic Polymers.

We developed a hybrid Monte Carlo self-consistent field technique to model physical gels composed of ABA triblock copolymers and gain insight into the structure and interactions in such gels. The associative A blocks of the polymers are confined to small volumes called nodes, while the B block can move freely as long as it is connected to the A blocks. A Monte Carlo algorithm is used to sample the node configurations on a lattice, and Scheutjens-Fleer self-consistent field (SF-SCF) equations are used to determine the change in free energy. The advantage of this approach over more coarse grained methods is that we do not need to predefine an interaction potential between the nodes. Using this MC-SCF hybrid simulation, we determined the radial distribution functions of the nodes and structure factors and osmotic compressibilities of the gels. For a high number of polymers per node and a solvent-B Flory-Huggins interaction parameter of 0.5, phase separation is predicted. Because of limitations in the simulation volume, we did however not establish the full phase diagram. For comparison, we performed some coarse-grained MC simulations in which the nodes are modeled as single particles with pair potentials extracted from SF-SCF calculations. At intermediate concentrations, these simulations gave qualitatively similar results as the MC-SCF hybrid. However, at relatively low and high polymer volume fractions, the structure of the coarse-grained gels is significantly different because higher-order interactions between the nodes are not accounted for. Finally, we compare the predictions of the MC-SCF simulations with experimental and modeling data on telechelic polymer networks from literature.

Encapsulation into complex coacervate core micelles promotes EGFP dimerization.

Complex coacervate core micelles (C3Ms) are colloidal structures useful for encapsulation of biomacromolecules. We previously demonstrated that enhanced green fluorescent protein (EGFP) can be encapsulated into C3Ms using the diblock copolymer poly(2-methyl-vinyl-pyridinium)41-b-poly(ethylene-oxide)205. This packaging resulted in deviating spectroscopic features of the encapsulated EGFP molecules. Here we show that for monomeric EGFP variant (mEGFP) micellar encapsulation affects the absorption and fluorescence properties to a much lesser extent, and that changes in circular dichroism characteristics are specific for encapsulated EGFP. Time-resolved fluorescence anisotropy of encapsulated (m)EGFP established the occurrence of homo-FRET (Förster resonance energy transfer) with larger transfer correlation times in the case of EGFP. Together, these findings support that EGFP dimerizes whereas the mEGFP mainly remains as a monomer in the densely packed C3Ms. We propose that dimerization of encapsulated EGFP causes a reorientation of Glu222, resulting in a pKa shift of the chromophore, which is fully reversible after release of EGFP from the C3Ms at a high ionic strength.

Complex coacervates formed across liquid interfaces: A self-consistent field analysis.

The Scheutjens-Fleer self-consistent field (SF-SCF) theory is used to study complexation between two oppositely charged polyelectrolytes across an interface formed by two solvents, here called oil and water. The focus is on the composition and the lateral stability of such interfacial coacervate. One polyelectrolyte is chosen to be oil soluble and the other one prefers water, whereas the counter and salt ions are taken to distribute ideally over all phases. There exists an electrostatic associative driving force for the formation of the coacervate phase which increases with decreasing ionic strength and may be assisted by some specific affinity between the associating units and an effective poor solvency for the coacervate. As with respect to the lateral stability an unusual wetting scenario, called pseudo-partial wetting, presents itself, which results from interactions on two different length scales. On the segmental length the screening of oil-water contacts promotes the wetting by the coacervate: a pre-wetting jump-like transition takes place off-coexistence from a microscopically thin to a mesoscopically thin film. Usually this implies complete wetting. However, the mesoscopically thin film is exposed to long-ranged attractive electrostatic interactions and therefore cannot grow to macroscopic dimensions upon approach towards coexistence. Hence the system remains partial wet. The bulk correlation length controls the thickness of the mesoscopically thin film and as a result the wetting transition occurs extremely close to the bulk critical point. We therefore expect that a thick coacervate film typically is laterally inhomogeneous: there are drops on top of a mesoscopically thin coacervate film. This conclusion qualitatively explains the experimental observation that such a coacervate film scatters visible light.

Characterisation of algal organic matter produced by bloom-forming marine and freshwater algae.

Algal blooms can seriously affect the operation of water treatment processes including low pressure (micro- and ultra-filtration) and high pressure (nanofiltration and reverse osmosis) membranes mainly due to accumulation of algal-derived organic matter (AOM). In this study, the different components of AOM extracted from three common species of bloom-forming algae (Alexandrium tamarense, Chaetoceros affinis and Microcystis sp.) were characterised employing various analytical techniques, such as liquid chromatography - organic carbon detection, fluorescence spectroscopy, fourier transform infrared spectroscopy, alcian blue staining and lectin staining coupled with laser scanning microscopy to indentify its composition and force measurement using atomic force microscopy to measure its stickiness. Batch culture monitoring of the three algal species illustrated varying characteristics in terms of growth pattern, cell concentration and AOM release. The AOM produced by the three algal species comprised mainly biopolymers (e.g., polysaccharides and proteins) but some refractory compounds (e.g., humic-like substances) and other low molecular weight acid and neutral compounds were also found. Biopolymers containing fucose and sulphated functional groups were found in all AOM samples while the presence of other functional groups varied between different species. A large majority (>80%) of the acidic polysaccharide components (in terms of transparent exopolymer particles) were found in the colloidal size range (<0.4 µm). The relative stickiness of AOM substantially varied between algal species and that the cohesion between AOM-coated surfaces was much stronger than the adhesion of AOM on AOM-free surfaces. Overall, the composition as well as the physico-chemical characteristics (e.g., stickiness) of AOM will likely dictate the severity of fouling in membrane systems during algal blooms.

On the edge energy of lipid membranes and the thermodynamic stability of pores.

To perform its barrier function, the lipid bilayer membrane requires a robust resistance against pore formation. Using a self-consistent field (SCF) theory and a molecularly detailed model for membranes composed of charged or zwitterionic lipids, it is possible to predict structural, mechanical, and thermodynamical parameters for relevant lipid bilayer membranes. We argue that the edge energy in membranes is a function of the spontaneous lipid monolayer curvature, the mean bending modulus, and the membrane thickness. An analytical Helfrich-like model suggests that most bilayers should have a positive edge energy. This means that there is a natural resistance against pore formation. Edge energies evaluated explicitly in a two-gradient SCF model are consistent with this. Remarkably, the edge energy can become negative for phosphatidylglycerol (e.g., dioleoylphosphoglycerol) bilayers at a sufficiently low ionic strength. Such bilayers become unstable against the formation of pores or the formation of lipid disks. In the weakly curved limit, we study the curvature dependence of the edge energy and evaluate the preferred edge curvature and the edge bending modulus. The latter is always positive, and the former increases with increasing ionic strength. These results point to a small window of ionic strengths for which stable pores can form as too low ionic strengths give rise to lipid disks. Higher order curvature terms are necessary to accurately predict relevant pore sizes in bilayers. The electric double layer overlap across a small pore widens the window of ionic strengths for which pores are stable.

Linking lipid architecture to bilayer structure and mechanics using self-consistent field modelling.

To understand how lipid architecture determines the lipid bilayer structure and its mechanics, we implement a molecularly detailed model that uses the self-consistent field theory. This numerical model accurately predicts parameters such as Helfrichs mean and Gaussian bending modulus kc and k̄ and the preferred monolayer curvature J(0)(m), and also delivers structural membrane properties like the core thickness, and head group position and orientation. We studied how these mechanical parameters vary with system variations, such as lipid tail length, membrane composition, and those parameters that control the lipid tail and head group solvent quality. For the membrane composition, negatively charged phosphatidylglycerol (PG) or zwitterionic, phosphatidylcholine (PC), and -ethanolamine (PE) lipids were used. In line with experimental findings, we find that the values of kc and the area compression modulus kA are always positive. They respond similarly to parameters that affect the core thickness, but differently to parameters that affect the head group properties. We found that the trends for k̄ and J(0)(m) can be rationalised by the concept of Israelachivili's surfactant packing parameter, and that both k̄ and J(0)(m) change sign with relevant parameter changes. Although typically k̄ < 0, membranes can form stable cubic phases when the Gaussian bending modulus becomes positive, which occurs with membranes composed of PC lipids with long tails. Similarly, negative monolayer curvatures appear when a small head group such as PE is combined with long lipid tails, which hints towards the stability of inverse hexagonal phases at the cost of the bilayer topology. To prevent the destabilisation of bilayers, PG lipids can be mixed into these PC or PE lipid membranes. Progressive loading of bilayers with PG lipids lead to highly charged membranes, resulting in J(0)(m) >> 0, especially at low ionic strengths. We anticipate that these changes lead to unstable membranes as these become vulnerable to pore formation or disintegration into lipid disks.

Bending moduli and spontaneous curvature of the monolayer in a surfactant bilayer.

We developed a method to evaluate the mechanical properties of the monolayers in symmetric surfactant bilayers using self-consistent field theory. A specific boundary condition is used to impose the same curvature onto the two opposing monolayers at the surfactant chemical potential equal to that of the corresponding homogeneously curved bilayer. Typically, the spontaneous monolayer curvature not equal 0 and its value depend on the surfactant architecture. This is of importance for the thermodynamics and topology of lamellar surfactant phases. Furthermore, it may be relevant in processes involving biological membranes, for example, the fusion and budding of vesicles and the incorporation of proteins in lipid bilayers.

Molecular modeling of lipid bilayers and the effect of protein-like inclusions.

We examined the effect of the insertion of a trans-membrane peptide or protein on the thermodynamic, structural and mechanical characteristics of a lipid bilayer using self-consistent field (SCF) theory. The peptide or protein is modeled as a rigid cylindrical body with a hydrophobic surface and two hydrophilic end caps. We first characterise the properties of the undisturbed bilayer consisting of lipids with a hydrophilic phosphatidylcholine-like head group of nine segments and two identical tails ranging from 8 to 19 alkyl segments. The structural properties are given in terms of thickness of the hydrophobic core d(l)0 and the area a0 per lipid. The mechanical or elastic properties are characterised by the mean bending modulus k(c), the saddle splay modulus k and the area compression-expansion modulus of the bilayer k(a) and its monolayers. Furthermore we calculated the spontaneous curvature J0(m) of the individual monolayer, which can be positive or negative depending on the tail length. Subsequently, we focus on the effect of hydrophobic mismatch between the bilayer and inclusion. The free energy of insertion is parabolic in the mismatch. The minimum is at a small negative mismatch, i.e., when the hydrophobic thickness of the rod is smaller than that of the bilayer. This is attributed to conformational restrictions of the lipid tails close to the rod. This results in a positive curvature of the bilayer adjacent to it, even if the hydrophobic thickness of the rod is larger than that of the bilayer. The bilayer deformation has a wave character which decays exponentially. We show that the decay length of this perturbation is the same as the elastic length (kc(d)2/ka)(1/4), provided that for the thickness d of the bilayer d(l)0 is used.

Double Layer of a Gold Electrode Probed by AFM Force Measurements.

Colloidal probe atomic force microscopy was used to determine the electric double layer interactions between a gold electrode and a spherical silica probe. The double layer properties of the gold/solution interface were varied through the pH and salt concentration of the electrolyte, as well as by externally applying an electric potential. The double layer potentials ψ(d) of the gold surface were obtained by fitting the force-distance curves according to the DLVO (Derjaguin-Landau-Verwey-Overbeek) theory, using earlier obtained values for the double layer potential of the silica probe as input parameter. It was found that the gold electrode combines the features of reversible and polarizable interfaces; i.e., its charge and potential are determined by both the solution pH and the external potential. The pH dependence is attributed to proton adsorption and desorption from oxidic groups on the gold surface. In the potential range studied, ψ(d) varies linearly with the applied potential; the variation in ψ(d) is roughly 10% of that in the applied potential. The potential of zero force (the external potential at which ψ(d) = 0) varies with pH. The various features of the gold/electrolyte interface are described well by an amphifunctional double layer model. The results of this study form the basis of the interpretation of adsorption studies on gold as a function of pH and externally applied potential.

Order in phospholipid Langmuir-Blodgett layers and the effect of the electrical potential of the substrate.

The ordering in dipalmitoylphosphatidylcholine (DPPC) Langmuir-Blodgett monolayers and bilayers on a semiconducting indium tin oxide (ITO) surface has been investigated at the equilibrium potential of the interface and at various externally applied potentials. Second- and fourth-rank order parameters of a diphenylhexatriene (DPH) containing phospholipid probe were derived from total internal reflection fluorescence measurements, and orientation distributions were calculated using the maximum-entropy method. Generally, bimodal orientation distributions were obtained, suggesting that only part of the probes is aligned with the DPPC molecules. The effect of applied potentials is small for DPPC layers on unmodified (hydrophilic) ITO; with decreasing potential the ordering changes slightly to more random distributions, possibly because of the onset of hydrogen evolution at the substrate surface. For monolayers on hydrophobized ITO, where the phospholipids are initially with their tails directed toward the surface, the changes are more significant. At the highest positive potential applied, the derived order parameters indicate that nearly all probes are flat on the surface. This can be understood as a result of enhanced competition between headgroups and tails for access to the surface as it becomes more polarized. On unmodified ITO the electrochemistry of Fe(CN)6(3-/4-) and Ru(bipyridyl)3(2+/3+) is hardly hindered by the presence of DPPC monolayers or bilayers. On hydrophobized ITO a DPPC monolayer enhances the redox reactions.

Order in phospholipid Langmuir-Blodgett monolayers determined by total internal reflection fluorescence.

Orientational order parameters of two diphenylhexatriene (DPH)-based fluorescent probes, 2-(3-(diphenylhexatrienyl)propanoyl)-1-hexadecanoyl-sn-glycero-3-p hosphocholine (DPHpPC) and 1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene (TMA-DPH), in dipalmitoylphosphatidylcholine (DPPC) Langmuir-Blodgett monolayers on quartz have been determined by total internal reflection fluorescence (TIRF). From these order parameters orientation distributions were reconstructed by the maximum-entropy method. For monolayers transferred from the liquid-condensed phase, preferential tilt angles with respect to the substrate normal around 14 degrees in the tail region and 5 degrees near the glycerol-acyl chain linkage were found, as reflected by the DPHpPC and TMA-DPH probes, respectively. The degree of ordering near the headgroup region seems to be larger than that further away from the surface. A substantial fraction of the TMA-DPH probes have a flat orientation and are probably located between the phospholipid headgroups and the substrate surface. Monolayers transferred from the liquid-expanded phase show a more random ordering, and most of the probe molecules (DPHpPC) are more or less flat on the surface. The results are consistent with earlier atomic force microscopy measurements on identical monolayers and are in reasonable agreement with previously published data on other organized phospholipid systems.

Determination of the orientation distribution of adsorbed fluorophores using TIRF. I. Theory.

The spectroscopic technique total internal reflection fluorescence can be used for determination of the orientation of adsorbed fluorescent molecules. The underlying theory is presented in general terms and elaborated in detail for the case that the fluorescent group is a porphyrin ring. It is shown that order parameters of the orientation distribution can be obtained if both the fluorescence intensity and its polarization are measured as functions of the polarization of the incident laser beam. From these order parameters an approximation of the orientation distribution can be derived by the maximum-entropy method.

Determination of the orientation distribution of adsorbed fluorophores using TIRF. II. Measurements on porphyrin and cytochrome c.

The theory for determination of the orientation of adsorbed fluorescent molecules using total internal reflection fluorescence, as explained in part I of this series, is illustrated by measurements on adsorbed tetramethylpyridinium porphyrin (H2TMPyP) and porphyrin cytochrome c molecules. The results are encouraging, although for porphyrin cytochrome c the scatter in the obtained order parameters is substantial. For H2TMPyP molecules adsorbed on glass the orientation distribution depends on the solution concentration. At low concentration, the H2TMPyP molecules are more or less randomly oriented, whereas at high concentrations a broad distribution around an angle of 46 degrees between the porphyrin plane and surface was found. For cytochrome c adsorbed on glass and indium tin oxide it was impossible to interpret the data in terms of orientation distributions because of the scatter in the results. The total fluorescence as a function of the polarization angle psi of the incident light beam corresponds to an average angle between the porphyrin group and the surface of 30 degrees-40 degrees. Despite the strong electric dipole moment of the protein, the orientation distribution seems to be independent on the (imposed) electrical potential of the interface.

Orientation of adsorbed cytochrome c as a function of the electrical potential of the interface studied by total internal reflection fluorescence.

A method for determination of the orientation of adsorbed structure-stable proteins using Total Internal Reflection Fluorescence is outlined. The theory has been elaborated for orientation studies on adsorbed free base cytochrome c, of which the prophyrin can be used as an intrinsic fluorescent label. The ratio of fluorescence intensities at two polarization modes of the incident light (the transverse magnetic and the transverse electric polarization mode, respectively) gives a relation between the orientation angles of the porphyrin relative to the interface. As an illustration of the theory, experimental results on the adsorption of cytochrome c at an optically transparent SnO2 film electrode are presented. It is concluded that the orientation of the molecules can only be affected by the interfacial potential during the process of adsorption, but, once adsorbed, the orientation cannot be changed anymore by variation of the potential.