Christine Kranz

Topography feedback mechanism for the scanning electrochemical microscope based on hydrodynamic forces between tip and sample

The distance between microelectrode and sample is a crucial parameter in scanning electrochemical microscopy. Here we report on a technique where the hydrodynamic coupling between tip and sample is employed to measure and control this distance. We built an apparatus, where a microelectrode is vibrated laterally and the damping of the amplitude upon approach to the surface is measured optically. We show that amplitudes of up to 5 μm are easily obtained in an aqueous environment and that amplitudes below 1 nm can be detected with phase sensitive amplifiers. We measured the different vibration modes of the electrodes and identified the ones best suited for distance measurements. On hard surfaces we found the characteristic decay length of the coupling to be less than 1 μm and, within our measured range, independent of frequency and amplitude of the vibration. Measurements on an elastic surface of known spring constant revealed that the vibrating electrode exerts a repulsive normal force on the sample which m...

Conducting polymer-based amperometric enzyme electrodes. Towards the development of miniaturized reagentless biosensors

Abstract The development of reagentless enzyme electrodes implies the covalent binding of enzymes and redox mediators on the sensor surface to prevent contamination of the sample by sensor components. Additionally, miniaturization and mass production of enzyme electrodes requires techniques for biosensor assembling avoiding manual deposition procedures. The electrochemical deposition of conducting polymer layers, e.g., polypyrrole, occurs at the surface of an electrode independent of its size and form. After the functionalization of conducting polymer films, electrode surfaces can be obtained which are suitable for the binding of enzymes or redox mediators. New reagentless enzyme electrodes can be obtained by electrochemical copolymerization of [Os(2,2′-bipyridien)2(3-{pyrrol-1-ylmethyl}pyridine)Cl]+], N-methylpyrrole and pyrrole-modified glucose oxidase leading to ternary copolymers simultaneously integrating redox relays and enzyme activity.

Topochemistry and photomechanical effects in crystals of green fluorescent protein-like chromophores: effects of hydrogen bonding and crystal packing.

To obtain insight into the effects of the environment on the photophysics and photochemistry of the green fluorescence protein (GFP), eight crystal structures of six synthetic aryl-substituted analogues (2-fluoro, 2-methyl, 3-hydroxy, 3-methoxy, 2,4-dimethyl and 2,5-dimethyl) of the GFP chromophore (4-hydroxy-benzylidenedimethylimidazolinone) were determined and correlated with their two-dimensional steady-state and time-resolved solid-state excitation-emission spectra. The stacking between the molecules greatly affected the emission energy and the lifetime of the emission of the chromophore, implying that pi-pi interactions could be critical for the photophysics of GFP. The reaction pathways were dependent on the excitation energy, resulting either in [2 + 2] photodimerization at the bridging double bond (UV excitation) or flipping of the imidazolone ring (visible excitation). The meta-hydroxy chromophore (3-HOBDI) was the only GFP-chromophore analogue that was obtained as more than one stable polymorph in the pure state thus far. Due to the asymmetric substitution with hydrogen bond donors and acceptors, 3-HOBDI is tetramorphic, the forms showing distinctly different structure and behavior: (1) while one of the polymorphs (3-HOBDI-A), having multilayer structure with alternating stereochemistry of linear hydrogen-bonded motifs, undergoes photodimerization under UV light, (2) another (3-HOBDI-C), which has dimeric head-to-tail structure, shows Z-to-E isomerization via tau-one-bond flip of the imidazolone ring by excitation in the visible region. X-ray diffraction analysis of a partially reacted single crystal of 3-HOBDI-C provided the first direct evidence of tau-one-bond flip occurring in a GFP-like compound. Moreover, the cooperative action of the photodimerization of 3-HOBDI-A appears as a photomechanical effect of unprecedented magnitude for a single crystalline specimen, where photoexcited single crystals bend to more than 90 degrees without breaking.

The role of conditioning film formation and surface chemical changes on Xylella fastidiosa adhesion and biofilm evolution

Biofilms are complex microbial communities with important biological functions including enhanced resistance against external factors like antimicrobial agents. The formation of a biofilm is known to be strongly dependent on substrate properties including hydrophobicity/hydrophilicity, structure, and roughness. The adsorption of (macro)molecules on the substrate, also known as conditioning film, changes the physicochemical properties of the surface and affects the bacterial adhesion. In this study, we investigate the physicochemical changes caused by Periwinkle wilt (PW) culture medium conditioning film formation on different surfaces (glass and silicon) and their effect on X. fastidiosa biofilm formation. Contact angle measurements have shown that the film formation decreases the surface hydrophilicity degree of both glass and silicon after few hours. Atomic force microscopy (AFM) images show the glass surface roughness is drastically reduced with conditioning film formation. First-layer X. fastidiosa biofilm on glass was observed in the AFM liquid cell after a period of time similar to that determined for the hydrophilicity changes. In addition, attenuation total reflection-Fourier transform infrared (ATR-FTIR) spectroscopy supports the AFM observation, since the PW absorption spectra increases with time showing a stronger contribution from the phosphate groups. Although hydrophobic and rough surfaces are commonly considered to increase bacteria cell attachment, our results suggest that these properties are not as important as the surface functional groups resulting from PW conditioning film formation for X. fastidiosa adhesion and biofilm development.

Imaging of microstructured biochemically active surfaces by means of scanning electrochemical microscopy

Abstract Scanning electrochemical microscopy (SECM) has been used to create and to image thin film structures of functionalized polypyrrole, to which glucose oxidase has been covalently immobilized. In addition, the SECM was applied to optimize the quality of all steps of this microfabrication process. For this purpose, imaging of the obtained structures was performed in the conventional feedback mode of the SECM with ferrocene derivatives or [Os (bpy) 2 fpy] (bpy = 2,2′-bipyridine, fpy = 4-formylpyridine). The same working mode is also used for the enzyme-modified microstructures exploiting the ability of glucose oxidase to accept the oxidized forms of these inorganic transition metal complexes as artificial cofactors. Successive images in the absence and presence of the enzymess substrate glucose added to the mediator solution allowed us to distinguish between the mediator regeneration caused by a heterogeneous electrontransfer reaction at the underlying gold support and the enzyme-mediated feedback. Prospects for the development of miniaturized biosensors are discussed.

FIB/SEM tomography with TEM-like resolution for 3D imaging of high-pressure frozen cells

Focused ion beam/scanning electron microscopy (FIB/SEM) tomography is a novel powerful approach for three-dimensional (3D) imaging of biological samples. Thereby, a sample is repeatedly milled with the focused ion beam (FIB) and each newly produced block face is imaged with the scanning electron microscope (SEM). This process can be repeated ad libitum in arbitrarily small increments allowing 3D analysis of relatively large volumes such as eukaryotic cells. High-pressure freezing and freeze substitution, on the other hand, are the gold standards for electron microscopic preparation of whole cells. In this work, we combined these methods and substantially improved resolution by using the secondary electron signal for image formation. With this imaging mode, contrast is formed in a very small, well-defined area close to the newly produced surface. By using this approach, small features, so far only visible in transmission electron microscope (TEM) (e.g., the two leaflets of the membrane bi-layer, clathrin coats and cytoskeletal elements), can be resolved directly in the FIB/SEM in the 3D context of whole cells.

Combined scanning electrochemical atomic force microscopy for tapping mode imaging

With the integration of submicro- and nanoelectrodes into atomic force microscopy(AFM) tips using microfabrication techniques, an elegant approach combining scanning electrochemicalmicroscopy (SECM) with atomic force microscopy has recently been demonstrated. Simultaneous imaging of topography and electrochemistry at a sample surface in AFM tapping mode with integrated SECM–AFM cantilevers oscillated at or near their resonance frequency is shown. In contrast to contact mode AFM imaging frictional forces at the sample surface are minimized. Hence, topographical and electrochemicalinformation of soft surfaces (e.g., biological species) can be obtained.

Integrating micro- and nanoelectrodes into atomic force microscopy cantilevers using focused ion beam techniques

This paper concerns a scanning probe capable of simultaneously measuring topography and local electrochemistry at a sample surface. Our approach ensures the distance regulation of the electrode by maintaining a fixed working distance between the probe and the sample surface independent from the electrochemical response. This is achieved by integrating micro- and nanoelectrodes into atomic force microscopy tips using focused ion beam techniques. The feasibility and functionality of the fully featured tip is demonstrated by a simultaneous topographical and electrochemical measurement of a porous polymer membrane as model surface.

Actin coating and compression of fused secretory vesicles are essential for surfactant secretion - a role for Rho, formins and myosin II

Secretion of vesicular contents by exocytosis is a fundamental cellular process. Increasing evidence suggests that post-fusion events play an important role in determining the composition and quantity of the secretory output. In particular, regulation of fusion pore dilation and closure is considered a key regulator of the post-fusion phase. However, depending on the nature of the cargo, additional mechanisms might be essential to facilitate effective release. We have recently described that in alveolar type II (ATII) cells, lamellar bodies (LBs), which are secretory vesicles that store lung surfactant, are coated with actin following fusion with the plasma membrane. Surfactant, a lipoprotein complex, does not readily diffuse out of fused LBs following opening and dilation of the fusion pore. Using fluorescence microscopy, atomic force microscopy and biochemical assays, we present evidence that actin coating and subsequent contraction of the actin coat is essential to facilitate surfactant secretion. Latrunculin B prevents actin coating of fused LBs and inhibits surfactant secretion almost completely. Simultaneous imaging of the vesicle membrane and the actin coat revealed that contraction of the actin coat compresses the vesicle following fusion. This leads to active extrusion of vesicle contents. Initial actin coating of fused vesicles is dependent on activation of Rho and formin-dependent actin nucleation. Actin coat contraction is facilitated by myosin II. In summary, our data suggest that fusion pore opening and dilation itself is not sufficient for release of bulky vesicle cargos and that active extrusion mechanisms are required.

Diamond-Modified AFM Probes: From Diamond Nanowires to Atomic Force Microscopy-Integrated Boron-Doped Diamond Electrodes

In atomic force microscopy (AFM), sharp and wear-resistant tips are a critical issue. Regarding scanning electrochemical microscopy (SECM), electrodes are required to be mechanically and chemically stable. Diamond is the perfect candidate for both AFM probes as well as for electrode materials if doped, due to diamonds unrivaled mechanical, chemical, and electrochemical properties. In this study, standard AFM tips were overgrown with typically 300 nm thick nanocrystalline diamond (NCD) layers and modified to obtain ultra sharp diamond nanowire-based AFM probes and probes that were used for combined AFM-SECM measurements based on integrated boron-doped conductive diamond electrodes. Analysis of the resonance properties of the diamond overgrown AFM cantilevers showed increasing resonance frequencies with increasing diamond coating thicknesses (i.e., from 160 to 260 kHz). The measured data were compared to performed simulations and show excellent correlation. A strong enhancement of the quality factor upon overgrowth was also observed (120 to 710). AFM tips with integrated diamond nanowires are shown to have apex radii as small as 5 nm and where fabricated by selectively etching diamond in a plasma etching process using self-organized metal nanomasks. These scanning tips showed superior imaging performance as compared to standard Si-tips or commercially available diamond-coated tips. The high imaging resolution and low tip wear are demonstrated using tapping and contact mode AFM measurements by imaging ultra hard substrates and DNA. Furthermore, AFM probes were coated with conductive boron-doped and insulating diamond layers to achieve bifunctional AFM-SECM probes. For this, focused ion beam (FIB) technology was used to expose the boron-doped diamond as a recessed electrode near the apex of the scanning tip. Such a modified probe was used to perform proof-of-concept AFM-SECM measurements. The results show that high-quality diamond probes can be fabricated, which are suitable for probing, manipulating, sculpting, and sensing at single digit nanoscale.