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A study on possible interactions between biomolecules and nanoparticlesKavianpour, Amir Houshang 22 October 2007
Along with the rapid growth of the nanotechnology, nanoparticles (NPs) have found many applications in commercial products. However, there are only a few studies on the toxicity and the environmental effects of NPs in biological systems. <p>In the study described in this thesis, I have used water-soluble Au NPs that were synthesized using the Brust method and then modified by small molecules. I explored the interactions of these modified Au NPs with self-assembled monolayer films on gold surfaces.<p>Three types of self-assembled monolayer (SAM) modified gold surfaces were used in this study. The surfaces had SAMs that could be positively or negatively charged or carry no charge, or be able to engage in hydrogen bonding. <p>Cyclic voltammetry (CV) was used to characterize SAMs of disulfide-glycine conjugate, disulfide-aspartic conjugate, and 11-mercaptoundecanoic acid (MUA) on gold surface electrodes. The possible interactions of Au NPs with the disulfide-aminoacid conjugates and alkanethiol modified surfaces were evaluated by cyclic voltammetry and by electrochemical impedance spectroscopy (EIS). An apparent decline in current density observed in CV along with an electron transfer resistance increase in EIS measurements upon exposure of the films to the MUA-modified anionic Au NPs clearly indicate interactions of the NPs with the films. Likewise, upon exposure of the films to cationic NPs, electron transfer resistance decreases dramatically in EIS experiments. In addition, the current increase in CV measurements provided further evidences for the interactions.
The interactions between modified Au NPs and the SAMs were investigated in more detail by infrared spectroscopy and by employing quartz crystal microbalance. These studies clearly showed that upon exposure of these SAM films to the water-soluble Au NPs, significant changes occur. As would be expected for the adsorption of the Au NPs onto the SAMs, the weight of the film increased due to the addition of the NPs on the surface. Moreover, there are significant increases in the carbonyl stretching vibration at 1735 cm-1 along with the appearance of the amide hydrogen stretching band, between 3160-3380 cm-1, which indicate the adsorption of Gly-CSA modified Au NPs onto the MUA film.
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Electron transfer mechanism and potential applications of α-helical peptidesMandal, Himadri Shekhar 26 October 2007
Understanding long range electron transfer (ET) in proteins is of fundamental interest to elucidate the complex nature of many biological processes. The mechanistic discussion is highly debated in the literature and the factors that control this process are still not clear. Because of the structural complexity and dynamic nature, it is very difficult to correctly evaluate long range ET in proteins. The study of simple model peptides having specific secondary structures is useful for a systematic and accurate evaluation. The polypeptide matrix in the photosynthetic reaction centre is rich in helices and this particular structural motif is believed to play an important role in ET in nature. In this thesis, ET study through some synthetic α-helical model peptides is described. The model peptides studied herein contain the redox-active ferrocene at one end and the thiol-functionalised Cys residue at the other. Films of these peptides were formed on the surface of gold electrodes via the Au-S bond, and by employing cyclic voltammetry, the rate of ET between the pendant ferrocene and the gold electrode through the peptide spacer has been evaluated. My study indicates that ET in α-helical peptides is a function of molecular dynamics and occurs via a tunnelling mechanism. These findings are significant and expected to offer new directions in the highly controversial discussion on ET in proteins.<p>This thesis also describes investigations in two important areas of applications of the α-helices. The first is photocurrent generation upon laser excitation of light-harvesting chromophore-functionalised peptides which mimics the natural photosynthetic centre. This important area of research can promote development of nano-scaled photovoltaic devices. Surprisingly, following the conventional experimental protocols, a photocurrent was observed in the absence of a chromophore and even by the irradiation of a bare gold electrode with laser light. It is suggested that an important consequence of laser irradiation has been overlooked in several publications and the so-called photocurrent phenomenon may be a consequence of laser heating. <p>Peptide-protected nanoparticles is another area of research receiving significant attention these days due to its potential relevance in biomedical applications. However, peptides are highly flexible and their structure can change depending on the nature of the environment. Since the reactivity of a peptide is related to its secondary structure, any conformational change could seriously alter the overall activity of the peptide-protected nanoparticles. In this thesis, the structural investigation of an α-helical peptide was carried out and it was found that the radius of curvature of nanoparticles has a profound effect on the structure of the adsorbate peptides and thereby, may affect the overall activity of the peptide-protected nanoparticles.
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Electrochemical neasurement of crevice corrosion of type AISI 304 stainless steelEtor, Aniekan 13 January 2010
Crevice corrosion is a form of galvanic corrosion that occurs when a metal is exposed to different environments. This occurs when the oxygen within the crevice gets depleted, thus acting as the anodic site for metal dissolution reaction. The anodic site thus encourages the migration of Cl- ions into the crevice leading to the development of an aggressive local solution. The acidic conditions present in the crevice reaches a critical crevice solution composition and results in the loss of stability of the passive film which further leads to a rapid breakdown of these films on the metal thus indicating the onset of active corrosion.<p>
In this research, it is hypothesized that the onset of crevice corrosion can be detected by measuring the galvanic coupling current between electrodes in a crevice and an external metal surface composed of the same material as the electrodes. To prove this hypothesis an engineered crevice was designed to measure IR controlled crevice currents along the crevice length of AISI 304 stainless steel immersed in a 0.5 M solution and a 1 M NaCl solution. Varying crevice openings were used to determine the effect of crevice gap (G) on the initiation of crevice corrosion and the position of the accelerated attack within the crevice.<p>
Multiplexed corrosion potential measurement and galvanic corrosion measurement techniques were used to measure the change in the open circuit potential (OCP) and the galvanic current for the four channels along the crevice length of the galvanic couple. The results obtained from the MGC test for the 100 µm crevice width immersed in 0.5 M NaCl solution showed good results with high anodic current at approximately 1 cm from the crevice mouth. This finding was in close agreement with the peak pH value observed at the position closest to the crevice mouth in the work of Alavi and Cottis (1987) and the model prediction of Kennell et al. 2009. However, for test samples with crevice width ≥ 200 µm, there was no initiation of crevice corrosion and the results obtained were discarded. The Linear polarization resistance scan and Potentiodynamic polarization scan carried out along the crevice to measure the polarization resistance, Rp , and to obtain the region of passivity along an AISI 304 SS crevice did not yield good results. Low corrosion rate in the range of 0.06 mm/yr was calculated for the AISI 304 stainless steel crevice.
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Electrochemical detection of interactions between DNA and various ligandsMuresan, Alina 04 December 2007
Antibodies specific for DNA, with varying degrees of sequence specificity, are common in many autoimmune diseases including systemic lupus erythematosus. The presence of anti-DNA antibodies is a useful determinant in arriving at a prognosis in these conditions. Given the prevalence of these diseases in both the developing and developed world and the difficulty that often accompanies diagnosis of autoimmune diseases, it is desirable to have sensitive, rapid, and inexpensive diagnostic tools for these diseases. Because of the great sensitivity of electrochemical techniques and their potential utility in characterizing interactions between macromolecules, electrochemistry has great potential as a diagnostic tool for any disease involving antibodies. Anti-DNA antibodies are present in many autoimmune diseases, notably systemic lupus erythematosus. Since DNA is a stable and well-characterized antigen, an electrochemical-based assay is particularly useful for diagnosis of these diseases. <p>The impedance of a gold surface was measured in the presence and absence of single- and double-stranded DNA monolayers. The DNA monolayer was diluted with butanethiol in order to provide a surface with more accessible binding sites than an undiluted monolayer. The change in impedance of the DNA monolayer following exposure to various small molecules and macromolecules was assessed. The molecules used included polyamines that induce conformational changes in DNA, proteins which bind DNA specifically, proteins which bind DNA non-specifically, and proteins which do not bind DNA. The presence of a DNA monolayer, whether single- or double-stranded, increased the impedance of the gold surface and dilution of the monolayers by butanethiol decreased the impedance, as expected. When exposed to polyamines, the impedance of the DNA monolayer decreased further. This could be due to lowered charge repulsion, to DNA condensation, or to a combination of both. When methylated bovine serum albumin was exposed to the monolayer, there was an increase in impedance. Conversely, when bovine serum albumin was exposed to the monolayer, the impedance was only increased at very high concentrations of protein. The increase following exposure to high concentrations of bovine serum albumin was likely due to deposition of protein on to the monolayer. The specificity of these interactions was illustrated by experiments with the antibody Hed 10, which binds single-stranded but not double-stranded DNA. Exposure to Hed 10 only caused a significant change in impedance when exposed to monolayers of single-stranded DNA.<p>The decreased impedance of the DNA monolayer caused by the presence of polyamines is consistent with the known structural perturbations induced by these molecules as measured with other methods. Similarly, the increase in impedance caused by the presence of proteins which bind DNA is consistent with increased steric interference by the protein-DNA complex. The failure of proteins which do not bind DNA to affect the impedance of the monolayer indicated that the effects in the experiments with DNA-binding proteins were due to protein binding and not other factors. The specificity of the assay as demonstrated by the results of the experiments with Hed 10 suggest that impedance-based measurements may provide the basis for a reliable, sensitive, and inexpensive assay for detecting the presence of anti-DNA antibodies in the serum of autoimmune disease patients.
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New concepts for electrical detection of biomoleculesDe la Rica Quesada, Roberto 17 September 2007 (has links)
Aquest treball discuteix difrerents aspects relacionats amb el disseny de sensors i sistemes de biodetecció. Descriu la fabricació i caracterització de transductors electrics particulars, així com el desenvolupament de nous sistemes de transduccio i el descobriment de noves methodologies per la fabricacio de nanomatrius de proteines.En primer lloc, es presenta un nou tipus de transductor impedimetric (I). Es va escollir un disseny basat en dos electrodes interdigitats per dos motius principals. Primer, aquesta geometria permet monitoritzar tant la resistència como la constant dieléctrica d'una solució, la qual cosa fa dels electrodes interdigitats eines més versatils que altres tipus transductors. Segon, els electrodes presenten una curta penetració del camp electric, la qual cosa els fa mes sensibles als canvis que tenen lloc a prop de la seva superfície. Aquest fet permet monitoritzar canvis locals en les magnituds d'interés. Finalment, són apropiats no nomes per construir sensors sinó també actuadors. Aquesta geometria sembla ser útil en experiments de dielectroforesi. Una innovació introduïda en aquesta tesi es el material escollit per fabricar els electrodes: silici policristal-lí o polisilici. El polisilici pot ser facilment modificat per donar lloc a superficies amb particulars propietats químiques i físiques, fent d'aquest material un excel-lent candidat per a la manufactura de biosensors, comparable a altres aproximacions com la quemisorció de alcanotiols sobre electrodes d'or. Els esmentats electrodes interdigitats es van fer servir per probar dos nous sistemes de transducció. Ambdues aproximacions comparteixen un tret comu: aprofiten la capacitat dels electrodes interdigitats per mesurar canvis local en les propietats elèctriques del medi on es troben submergits. En II, aquest fet és utilitzat per monitoritzar una reacció enzimàtica, i es mostra com la característica de mesura local en electrodes interdigitats dóna lloc a una detecció més sensible. A més, es demostra que aquesta aproximació es adequada per la detecció de proteïnes fent servir l'enzim com a marca en un immunoassaig. En III, els electrodes interdigitats actuen com a sensor i actuador. Com a actuador, els electrodes son capaços de concentrar esferes de làtex a la seva superficie. Com a transductors, la presencia de les micropartícules aïllants a la seva superficie dóna lloc a un canvi en la geometria de la cel-la, que pot ser detectat monitoritzant tant la resistència com la capacitat de la solucio. Aquest mode de funcionament es paral-lel al dels sensors magnetoresistius, i el principi de transduccio proposat es presenta com a una alternativa a ells.Finalment, un quart treball es presenta en aquesta tesi (anex). Comparteix dues característiques en comú amb els treballs previs: el sustrat (silici) i una metodologia per la inmoblització de biomolecules (silanització). Les seves aplicacions son, però, diferents i cobreixen un rang més ampli d'aplicacions. En concret, una nova metodologia pel nanoestructurat de superfícies, de baix cost i fàcil disponibilitat és presentada. Es van aconseguir motius fets amb molècules de silà amb dimensions inferiors als 10 nm. En el marc de la biodetecció, aquesta nova tècnica per nanoestructurat superficial es propossa com a alternativa a la nanolitografia dip-pen per la manufactura de nanomatrius de biomolècules. Les petites dimensions dels motius obtinguts obren el cami per la consecució de nanomatrius d'una única molècula. / This work discusses different aspects related to the design of biosensors and biodetection systems. It describes the fabrication and characterization of particular electric transducers together with the development of new transduction systems and the finding of new methodologies for biomolecule nanoarray fabrication.Firstly, a new type of impedimetric transducer is presented (I). A two-electrode interdigitated design was chosen, mainly for three reasons. First, this geometry allows the monitoring of both the resistivity and the dielectric constant of a solution, thus making interdigitated electrodes more versatile tools than other kind of transducers. Second, they present short electric field penetration depths, which make them more sensitive to changes occurring close to their surface. This fact enables the monitoring of local changes in the magnitudes of interest. Finally, they are suitable for constructing not only sensors but also actuators. This geometry appears to be useful in dielectrophoresis experiments. One innovation introduced in this thesis is the material chosen to fabricate the electrodes: polycrystalline silicon, also known as polysilicon. Polysilicon can be easily modified to render surfaces with distinct physical and chemical properties, thus making this material an excellent approach for biosensors manufacture, comparable to other approaches like alkanethiol chemisorption on gold electrodes. The aforementioned interdigitated electrodes were used to test two new transduction principles. The two approaches share a common feature: they rely on the ability of interdigitated electrodes to measure local changes in the electrical properties of the medium where they are immersed. In II, this is used to monitor an enzymatic reaction, and it is shown that the characteristics of measuring local changes at interdigitated electrodes result in a more sensitive detection. Furthermore, the feasibility of this approach for protein detection is demonstrated by using the enzyme as a label for performing an immunoassay. In III, the interdigitated electrodes act both as a transducer and as an actuator. As an actuator, the electrodes are able to concentrate latex beads at their surface. As a transducer, the presence of the insulating microparticles at their surface results in a change in the geometry of the cell, that can be detected by monitoring either the resitance or the capacitance of the solution. Such device performance is parallel to that of magnetoresistive biosensors, and the proposed transduction principle is envisaged as a suitable alternative to them.Finally, a fourth work is presented in this thesis (Annex). It shares two features in common with the previous works: the substrate (silicon) and a method for biomolecule immobilization (silanization). However, the applications are somehow different, and cover a wider range. Precisely, a new methodology for low cost, easily available nanopatterning is shown. Features made of silane molecules, with dimensions less than 10 nm are successfully patterned. In the frame of biodetection, this new nanopatterning technique is proposed as an alternative to dip-pen nanolithography in nanoarray manufacture. Moreover, the small dimensions of the obtained patterns pave the way for the achievement of single-molecule nanoarrays.
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Nanoprecipitation in Quartz Nanopipettes and Application in the Crystallization of Inorganic SaltsBrown, Warren D 07 August 2012 (has links)
The high surface to volume ratio which is a property of nanoscale devices means the interfacial effects from these devices on the mass transport of analyte can be significant. Quartz nanopipette effect on the mass transport behavior of inorganic monovalent salts such as potassium chloride is shown to differ from those of conical nanopore. Quartz nanopipettes demonstrate a more significant interfacial impact on the mass transport behavior of inorganic salts. This is evidenced by significant impacts on ionic transport even at high electrolyte concentration where nanopore interfacial effects do not significantly impact the ion transport. Nanopipettes have been use to precipitate salts such as lithium chloride in bulk concentrations three orders of magnitude below the saturation concentration. These novel interfacial interactions have opened new avenues for crystallization of more complex organic biomolecules using inorganic systems as model systems on which to base the approach for these more complex systems.
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Development of methods for determination of adsorption kinetics at metal electrodesMoyana, Agata 01 January 1996 (has links)
Adsorption at metal electrodes is usually a very fast process and it plays a most important role in many areas of industry. The thermodynamics of the process are well known for many systems. However, there is currently no good method that allows a determination of very fast kinetics of adsorption to be made. Previously, many attempts at evaluation of kinetic parameters of adsorption were made, but in moat cases, due to the inadequacy of the experimental methods used, the parameters obtained were much lower than expected. This thesis aims at providing the means for determining the kinetics of adsorption at metal electrodes. The methods herein described are based on two different experimental techniques. These techniques are: (i) fast cyclic voltammetry (FCV, potential sweep rate up to 100000 V/s) and (ii) high frequency AC and FFT SW (Fast Fourier Transform Square-wave) voltammetry (frequency up to 50 MHz) at ultramicroelectrodes (5 or 6.25 ìm in radius). A theoretical description of the adsorption process for both kinds of experiments is presented. A simulation program was written to provide a better understanding of the process and to elucidate the development of methods for determining the kinetics of adsorption. Thermodynamic and kinetic descriptions of the process are based on the Frumkin adsorption isotherm. Both the equilibrium constant and the adsorption rate constant are treated as functions of potential and the electrode coverage. Comparison of results for different systems is presented as an analysis of the dependence of the adsorption rate constant on the equilibrium constant. FCV proved to be useful in the evaluation of kinetics of chemisorption (standard rate constant in the range of 10<sup>6</sup> s$\sp{-1})$ but the results for adsorption of aliphatic alcohols were unreliable. High frequency AC methods allowed the determination of kinetics of physical adsorption. It was found that the activation energy of the adsorption process can be expressed as a linear combination of the electrical component of the standard free energy of adsorption (a major contribution) and the energy of lateral interactions (a minor contribution). At the zero charge potential the rate constant reaches the maximum value of $\rm(4.6\pm0.3)10\sp9\ s\sp{-1}.$
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Electrochemical determination of surface active compounds at noble metal ultramicroelectrodes in flowing solutionsNorouzi, Parviz 01 January 1999 (has links)
In this work, a new electrochemical detection method was developed with the ability to determine a wide range of inorganic and organic species at, trace levels. In brief, the detection method takes advantage of all possible electrochemical reactions that may occur during scanning of the electrode potential. Changes in the detector response are mainly the result of inhibition of oxygen adsorption and hydrogen adsorption, alteration of electrical properties of the double layer, or redox processes of the adsorbate. Various electrochemical techniques were examined in the measurements; i.e. cyclic voltammetry, pulse amperometric detection, and square wave voltammetry. In those electrochemical techniques, the detection was carried out in a stripping mode after accumulation of analytes on the electrode surface. The smallest discernable signal is associated with about 0.1% surface coverage, which corresponds to the adsorption of about 10-18 mol of analyte on a ultramicroelectrode 5-[mu]m in radius. The response time of the detector to the concentration change in most cases is less than 1s. Electrochemical conditioning of the working electrode is sufficient to ensure a stable response for a period of several hours. It appears that square wave and cyclic voltammetry techniques are more suitable for the detection method. The linear dynamic range of the calibration curve depends on the characteristic of the analyte-electrode bond and redox processes of the analyte, which may occur at the electrode surface. For instance, for strongly adsorbing molecules the linear dynamic range extends over two orders of magnitude from about 10 -7 M to 10-5 M and for electroactive compounds from about 10-8 M to 10-4 M. In general, the relative standard deviation for replicate determinations was lower than 5%. Moreover, in these analyses, removal of oxygen from the analyzed solutions is not required.
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Electrochemical detection of interactions between DNA and various ligandsMuresan, Alina 04 December 2007 (has links)
Antibodies specific for DNA, with varying degrees of sequence specificity, are common in many autoimmune diseases including systemic lupus erythematosus. The presence of anti-DNA antibodies is a useful determinant in arriving at a prognosis in these conditions. Given the prevalence of these diseases in both the developing and developed world and the difficulty that often accompanies diagnosis of autoimmune diseases, it is desirable to have sensitive, rapid, and inexpensive diagnostic tools for these diseases. Because of the great sensitivity of electrochemical techniques and their potential utility in characterizing interactions between macromolecules, electrochemistry has great potential as a diagnostic tool for any disease involving antibodies. Anti-DNA antibodies are present in many autoimmune diseases, notably systemic lupus erythematosus. Since DNA is a stable and well-characterized antigen, an electrochemical-based assay is particularly useful for diagnosis of these diseases. <p>The impedance of a gold surface was measured in the presence and absence of single- and double-stranded DNA monolayers. The DNA monolayer was diluted with butanethiol in order to provide a surface with more accessible binding sites than an undiluted monolayer. The change in impedance of the DNA monolayer following exposure to various small molecules and macromolecules was assessed. The molecules used included polyamines that induce conformational changes in DNA, proteins which bind DNA specifically, proteins which bind DNA non-specifically, and proteins which do not bind DNA. The presence of a DNA monolayer, whether single- or double-stranded, increased the impedance of the gold surface and dilution of the monolayers by butanethiol decreased the impedance, as expected. When exposed to polyamines, the impedance of the DNA monolayer decreased further. This could be due to lowered charge repulsion, to DNA condensation, or to a combination of both. When methylated bovine serum albumin was exposed to the monolayer, there was an increase in impedance. Conversely, when bovine serum albumin was exposed to the monolayer, the impedance was only increased at very high concentrations of protein. The increase following exposure to high concentrations of bovine serum albumin was likely due to deposition of protein on to the monolayer. The specificity of these interactions was illustrated by experiments with the antibody Hed 10, which binds single-stranded but not double-stranded DNA. Exposure to Hed 10 only caused a significant change in impedance when exposed to monolayers of single-stranded DNA.<p>The decreased impedance of the DNA monolayer caused by the presence of polyamines is consistent with the known structural perturbations induced by these molecules as measured with other methods. Similarly, the increase in impedance caused by the presence of proteins which bind DNA is consistent with increased steric interference by the protein-DNA complex. The failure of proteins which do not bind DNA to affect the impedance of the monolayer indicated that the effects in the experiments with DNA-binding proteins were due to protein binding and not other factors. The specificity of the assay as demonstrated by the results of the experiments with Hed 10 suggest that impedance-based measurements may provide the basis for a reliable, sensitive, and inexpensive assay for detecting the presence of anti-DNA antibodies in the serum of autoimmune disease patients.
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Peptide monolayers : an electrochemical studyOrlowski, Grzegorz Artur 05 September 2007 (has links)
Understanding electron-transfer (ET) processes in proteins is of fundamental importance. In a series of photophysical studies of well-behaved peptide model systems, it has become evident that the ET through peptide spacers is greatly influenced by the separation between the acceptor (A) and the donor (D), the nature of the peptide backbone, the amino acid sequence, and the resulting flexibility of the peptide conjugates. In particular, it was suggested in the literature that the presence of H-bonding will increase the rate of ET, and there is experimental evidence, mostly in proteins, to suggest that H-bonding indeed increases the rate of ET.<p>My aim was to develop a potential-assisted deposition method for ferrocene peptide disulfides onto gold surfaces and investigate the electrochemical properties of these films. We made use of two classes of Fc-peptides: acylic ferrocenoyl (Fc)-peptide disulfides and cyclo-1,1-Fc-peptide disulfides, allowing the preparation of tightly packed films of cyclic and acylic Fc-peptides on gold surfaces within 30 minutes. This is a significant benefit compared to the conventional soaking method of self-assembly requiring several days for the assembly of well-packed films. Such films exhibited considerably improved stability. This electrodeposition method should find wide-spread applications for the formation of tightly-packed films from disulfides. Our studies allowed a direct comparison of the electron transfer kinetics of cyclic and acyclic Fc-peptide disulfide systems. Our results showed faster ET kinetics for films prepared from cyclic Fc-peptide conjugates compared to the acyclic systems, presumably as a result of the enhanced rigidity of the Fc-peptide conjugates on the surface and/or an increase of the number of conductive peptide wires to the surface. Following the idea of peptide dynamics as a major contributor to the observed electron transfer rate in peptides and peptide conjugates, variable temperature electrochemical studies of Fc-peptide films were performed. An estimation of the reorganization energy associated with ferrocene/ferrocenium (Fc/Fc+) redox process allowed us to probe the role of peptide dynamics. Three counter-ions were tested, exhibiting different strengths of association with the Fc+ group (BF4- < ClO4- < PF6-) and the reorganization energies were evaluated in each case. The highest reorganization energy was obtained for the weakly interacting anion BF4-. Weakly interacting anions also showed significant broadness in the redox peaks and emergence of the second oxidation peak which is attributed to phase separation of the ferrocene group. Ferrocene agglomeration was not observed for any of the cyclic Fc-peptide conjugates but occurred for some of the acyclic systems. In particular, for acyclic Val and Leu containing Fc-peptide conjugates agglomeration were observed and was presumably caused by lateral interactions between the hydrophobic side-groups of the peptides. Further experiments involving the interaction of Fc-peptide films with alkali metal ions gave additional evidence that electron transfer is influenced significantly by peptide dynamics.
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