Dan Bizzotto

On the nature of DNA self-assembled monolayers on Au: measuring surface heterogeneity with electrochemical in situ fluorescence microscopy.

The creation of gold surfaces modified by single- or double-stranded DNA self-assembled monolayers (SAMs) is shown to produce heterogeneous surface packing densities through the use of electrochemical studies coupled with fluorescence imaging. The modified surfaces created by direct adsorption of thiolate DNA [followed by passivation with mecaptohexanol (MCH)] resulted in regions covered by a monolayer of DNA SAM and other regions that were coated by large particles of DNA. The difference in fluorescence intensity measured from these regions was dramatic. More importantly, a regional variance in fluorescence intensity in response to electrochemical potential was observed: the large aggregates showing a significantly different modulation of fluorescence intensity than the monolayer-coated regions. Electrochemical desorption and detection of the fluorescently tagged DNA provided clear evidence of a complete surface modification. These studies have implications for biosensor/biochip development using DNA SAMs. A modification in the method used to produce the DNA SAMs resulted in a significantly different surface with much fewer aggregates and more significant electromodulation of the fluorescence intensity, though at much lower DNA surface density (ca. 1% of maximum theoretical coverage). This method for forming the modified surfaces has clear advantages over the currently accepted practice and emphasizes the importance of studying the nonaveraged nature of the sensor surface using in situ imaging tools like electrofluorescence microscopy.

Chloride adsorption at the Au(111) electrode surface

The adsorption of iodide at a Au(111) single crystal electrode has been investigated quantitatively using chronocoulometry. By analyzing the charge density data thermodynamically, the following parameters were determined: the Gibbs excess, Gibbs energy of adsorption, the number of electrons flowing to the interface per one adsorbed iodide ion at a constant electrode potential (electrosorption valency), and at a constant chemical potential. The thermodynamic data for iodide adsorption were compared to the results for bromide and chloride adsorption. All the three halides form a chemisorption bond with the gold surface. The bond is quite polar at the negatively charged surface, however, its polarity drops significantly at the positively charged surface. At low charge densities and coverages, the bond polarity is determined by the ability of free electrons to screen the dipole formed by the adsorbed anion and its image charge in the metal. At high charge densities and coverages, the chemisorption bond has a predominantly covalent character. The strength of the halide adsorption and the covalent character of the chemisorption bond increase progressively by moving from chloride to iodide.

Fluorescence imaging of the oxidative desorption of a BODIPY-alkyl-thiol monolayer coated Au bead.

The reductive and oxidative desorption of a BODIPY labeled alkylthiol self-assembled monolayer (SAM) on Au was studied using electrochemical methods coupled with fluorescence microscopy and image analysis procedures to monitor the removal of the adsorbed layer. Two SAMs were formed using two lengths of the alkyl chain (C10 and C16). The BODIPY fluorescent moiety used is known to form dimers which through donor-acceptor energy transfer results in red-shifted fluorescence. Fluorescence from the monomer and dimer were used to study the nature of the desorbed molecules during cyclic step changes in potential. The reductive desorption was observed to occur over a small potential window (0.15 V) signified by an increase in capacitance and in fluorescence. Oxidative readsorption was also observed through a decrease in capacitance and a lack of total removal of the fluorescent layer. Removal by oxidative desorption occurred at positive potentials over a broad potential range near the oxidation of the bare Au. The resulting fluorescence showed that the desorbed molecules remained near the electrode surface and were not dispersed over the 20 s waiting time. The rate of change of the fluorescence for oxidative desorption was much slower than the reductive desorption. Comparing monomer and dimer fluorescence intensities indicated that the dimer was formed on the Au surface and desorbed as a dimer, rather than forming from desorbed monomers near the electrode surface. The dimer fluorescence can only be observed through energy transfer from the excited monomer suggesting that the monomers and dimers must be in close proximity in aggregates near the electrode. The fluorescence yield for longer alkyl chain was always lower presumably due to its decreased solubility in the interfacial region resulting in a more efficient fluorescence quenching. The oxidative desorption process results in a significantly etched or roughened electrode surface suggesting the coupling of thiol oxidative removal and Au oxide formation which results in the removal of Au from the electrode.

Adsorption of DOPC onto Hg from the G ∣ S interface and from a liposomal suspension

Abstract The adsorption of various physical forms of dioleoyl phosphatidylcholine (DOPC) onto a Hg electrode have been studied. The three systems investigated were adsorption of DOPC from the monolayer state present on the gasxa0∣xa0solution (Gxa0∣xa0S) interface, the adsorption of 100 nm DOPC liposomes present in the electrolyte, and a mixed system consisting of the co-adsorption of the monolayer and liposomes. The adsorption was characterized by capacitance measurements, chronoamperometry and impedance spectroscopy. The adsorbed layer produced by liposome adsorption was distinctly different as compared to the layer adsorbed from the Gxa0∣xa0S interface, whereas the co-adsorbed layer retained most of the liposome layer qualities. A low capacitance region, centered at −0.40 V (SCE) with values of 1.85, and 1.45 μF cm−2 for the Gxa0∣xa0S and the liposome system were, respectively observed. This region was stable with respect to very negative potential cycling only for the liposome systems. Three phase transitions (denoted by pseudo-capacitance peaks) were noted for the three systems under study; two corresponding to a change in the adsorbed state, and one corresponding to the adsorption–desorption process. The kinetics of these changes were quite different and depended on the nature of the adsorbing species. The most significant differences were observed when measuring the capacity over a large range of perturbation frequency. At low frequencies, desorption of the DOPC layer was observed at very negative potentials for all the systems studied. As the perturbation frequency was increased, the capacitance measured for the Gxa0∣xa0S system maintained this characteristic, but surprisingly this was not the case for the liposome systems. In both cases (liposome and co-adsorbed) the capacity, calculated assuming a series RC circuit, was found to be significantly lower than the capacity measured for the Hgxa0∣xa00.1 M KCl interface. Preliminary impedance spectra are presented illustrating the non-ideal behavior of the liposomal system. This phenomenon is undergoing further study. A possible mechanism for the adsorption of DOPC from the liposomal state is also presented.

Electrochemical and spectroscopic studies of the mechanism of monolayer and multilayer adsorption of an insoluble surfactant at the Au(111) | electrolyte interface

Abstract The adsorption of an insoluble monolayer of 12-(9-anthroyloxy) stearic acid (12-AS) onto a single crystal gold electrode has been described. The spreading of the insoluble surfactant onto the metal | solution interface of the gold electrode was initially investigated with the help of cyclic voltammetry and differential capacity. Electrochemical studies indicate that the film of 12-AS molecules spreads onto the metal surface at potentials close to the potential of zero charge (pzc) and desorbs from the electrode surface at potentials that are sufficiently negative. The spreading of the film and its subsequent desorption are repeatable. The 12-AS molecule is a surfactant dye which may be used to perform spectroelectrochemical experiments. Electroreflectance spectroscopy, fluorescence spectroscopy and measurements of the light scattered by the desorbed surfactant molecules were employed to determine the mechanism by which molecules of the insoluble surfactant spread onto and desorb from the metal | solution interface of the gold electrode. With the help of the spectroelectrochemical experiments we have demonstrated that the repeatable potential-induced desorption and adsorption (spreading) of the insoluble molecules involves formation of micelles (or similar molecular organizations) in the subsurface region and spreading of the micelles onto the electrode surface.

Epi-fluorescence microscopic characterization of potential-induced changes in a DOPC monolayer on a Hg drop

Characterization of the potential-induced changes of a lipid-coated Hg-0.1 M KCl interface through electrochemical techniques and newly developed in situ fluorescence microscopy is described. Fluorescence of a fluorophore-containing dioleoyl phosphatidylcholine (DOPC) layer deposited from the gas-solution interface was observed to be dependent upon the potential of the Hg surface. The largest changes occurred for potentials where the lipid layer was desorbed: the lipid moved away from the electrode surface, reducing the efficiency of metal-mediated quenching of the excited state resulting in an increase in fluorescence. Electric potential-induced changes in the morphology of the adsorbed or desorbed DOPC lipid monolayer were observed optically for the first time using this technique. The observed potential-dependent fluorescence was compared to previous studies on an octadecanol-coated Au(111) electrode. Fluorescence microscopy was also used to characterize the fusion of DOPC liposomes with a previously adsorbed DOPC layer. Large changes in fluorescence were observed for the DOPC layer after fusion with liposomes. The fusion was accomplished via potential-created defects in the adsorbed DOPC monolayer through which the liposomes interact. The integration of the liposomes into the adsorbed monolayer results in a hybrid layer in which some lipid exists further from the electrode surface, resulting in a large increase in fluorescence. Possibilities for the creation of a biomimetic adsorbed hybrid lipid layer on Hg are also discussed.

Influence of Surface Structure on Single or Mixed Component Self-Assembled Monolayers via in Situ Spectroelectrochemical Fluorescence Imaging of the Complete Stereographic Triangle on a Single Crystal Au Bead Electrode

The use of a single crystal gold bead electrode is demonstrated for characterization of self-assembled monolayers (SAM)s formed on the bead surface expressing a complete set of face centered cubic (fcc) surface structures represented by a stereographic projection. Simultaneous analysis of many crystallographic orientations was accomplished through the use of an in situ fluorescence microscopic imaging technique coupled with electrochemical measurements. SAMs were prepared from different classes of molecules, which were modified with a fluorescent tag enabling characterization of the influence of electrical potential and a direct comparison of the influence of surface structure on SAMs adsorbed onto low index, vicinal and chiral surfaces. The assembly of alkylthiol, Aib peptide and DNA SAMs are studied as a function of the electrical potential of the interface revealing how the organization of these SAMs depend on the surface crystallographic orientation, all in one measurement. This approach allows for a simultaneous determination of SAMs assembled onto an electrode surface onto which the whole fcc stereographic triangle can be mapped, revealing the influence of intermolecular interactions as well as the atomic arrangement of the substrate. Moreover, this method enables study of the influence of the Au surface atom arrangement on SAMs that were created and analyzed, both under identical conditions, something that can be challenging for the typical studies of this kind using individual gold single crystal electrodes. Also demonstrated is the analysis of a SAM containing two components prepared using thiol exchange. The two component SAM shows remarkable differences in the surface coverage, which strongly depends on the surface crystallography enabling estimates of the thiol exchange energetics. In addition, these electrode surfaces enable studies of molecular adsorption onto the symmetry related chiral surfaces since more than one stereographic triangle can be imaged at the same time. The ability to observe a SAM modified surface that contains many complete fcc stereographic triangles will facilitate the study of the single and multicomponent SAMs, identifying interesting surfaces for further analysis.

Effect of Heat-Treated Amphotericin B on Renal and Fungal Cytotoxicity

ABSTRACT The purpose of this investigation was to determine the cytotoxicity of amphotericin B (AMB; trade name Fungizone [FZ]) following the administration of FZ and a heat-treated form of FZ (HFZ) to LLC-PK1 pig kidney cells and Cryptococcus neoformans var. gattii cells. HFZ was significantly less toxic to kidney cells than FZ at all concentrations tested. For both FZ and HFZ, the concentration range which resulted in a 50% reduction of the growth of fungal cells was 0.125 to 1 mg/ml. These findings suggest that heat treatment decreases AMBs renal cytotoxicity without modifying its antifungal activity.

Abnormal Grain Growth in Electrochemically Deposited Cu Films

Cu interconnects are essential in advanced integrated circuits to minimize the RC delay. In manufacturing these devices, Cu is deposited electrochemically using a plating bath containing organic additives. The as-deposited nanocrystalline Cu films undergo self-annealing at room temperature to form a micronsized grain structure by abnormal grain growth. Systematic experimental studies of self-annealing kinetics on model Cu films deposited on a Au substrate suggest that the rate of grain size evolution depends primarily on the initial grain size of the asdeposited film. A model for the observed abnormal grain growth process is proposed. Assuming that desorption of the organic additives leads to mobile grain boundaries, the onset of abnormal grain growth is attributed to a sufficiently low additive concentration such that a full coverage of all grain boundaries cannot be maintained. The incubation time of abnormal growth is then a logarithmic function of the initial grain size. The probability to find a growing grain is proportional to the number of grains per unit volume. This assumption is seen to be in good agreement with the experimental observations for subsequent abnormal grain growth rates. The limitations of the proposed model and the challenges to obtain further insight into the complex microstructure mechanisms during self-annealing are delineated.

What happens to the thiolates created by reductively desorbing SAMs? An in situ study using fluorescence microscopy and electrochemistry.

In situ examination of the reductive desorption process for Au microelectrodes modified with a thiol self-assembled monolayer (SAM) using fluorescence microscopy enabled the study of the fate of the desorbed thiolate species. The Bodipy labeled alkyl-thiol SAM, when adsorbed, is not fluorescent due to quenching by the Au surface. Once reductively desorbed, the thiolate molecules fluoresce and their direction and speed are monitored. At moderately negative reduction potentials, the thiolate species hemispherically diffuse away from the microelectrode. Also observed is the influence of a closely positioned counter electrode on the direction of the desorbed thiolate movement. As the potential becomes more negative, the molecules move in an upward direction, with a speed that depends on the amount of dissolved H(2) produced by water reduction. Shown is that this motion is controlled, in large part, by the change in the electrolyte density near the electrode due to dissolved H(2). These results should help in explaining the extent of readsorption at oxidative potentials observed in cyclic voltammetry (CV) reductive desorption measurements, as well as improving the general understanding of the SAM removal process by reductive desorption. The electrogenerated H(2) was also shown to be able to reductively remove the thiol SAM from the Pt/Ir particles that decorate the microelectrode glass sheath.