Dorothea Brüggemann

Nanostructured gold microelectrodes for extracellular recording from electrogenic cells

We present a new biocompatible nanostructured microelectrode array for extracellular signal recording from electrogenic cells. Microfabrication techniques were combined with a template-assisted approach using nanoporous aluminum oxide to develop gold nanopillar electrodes. The nanopillars were approximately 300-400 nm high and had a diameter of 60 nm. Thus, they yielded a higher surface area of the electrodes resulting in a decreased impedance compared to planar electrodes. The interaction between the large-scale gold nanopillar arrays and cardiac muscle cells (HL-1) was investigated via focused ion beam milling. In the resulting cross-sections we observed a tight coupling between the HL-1 cells and the gold nanostructures. However, the cell membranes did not bend into the cleft between adjacent nanopillars due to the high pillar density. We performed extracellular potential recordings from HL-1 cells with the nanostructured microelectrode arrays. The maximal amplitudes recorded with the nanopillar electrodes were up to 100% higher than those recorded with planar gold electrodes. Increasing the aspect ratio of the gold nanopillars and changing the geometrical layout can further enhance the signal quality in the future.

Nanoporous aluminium oxide membranes as cell interfaces

Nanoporous anodic aluminium oxide (AAO) has become increasingly important in biomedical applications over the past years due to its biocompatibility, increased surface area, and the possibility to tailor this nanomaterial with a wide range of surface modifications. AAO nanopores are formed in an inexpensive anodisation process of pure aluminium, which results in the selfassembly of highly ordered, vertical nanochannels with well-controllable pore diameters, depths, and interpore distances. Because of these outstanding properties AAO nanopores have become excellent candidates as nanostructured substrates for cell-interface studies. In this comprehensive review previous surveys on cell adhesion and proliferation on different AAO nanopore geometries and surface modifications are highlighted and summarised tabularly. Future applications of nanoporous alumina membranes in biotechnology and medicine are also outlined, for instance, the use of nanoporous AAO as implant modifications, coculture substrates, or immunoisolation devices.

A nanoporous alumina microelectrode array for functional cell?chip coupling

The design of electrode interfaces has a strong impact on cell-based bioelectronic applications. We present a new type of microelectrode array chip featuring a nanoporous alumina interface. The chip is fabricated in a combination of top-down and bottom-up processes using state-of-the-art clean room technology and self-assembled generation of nanopores by aluminum anodization. The electrode characteristics are investigated in phosphate buffered saline as well as under cell culture conditions. We show that the modified microelectrodes exhibit decreased impedance compared to planar microelectrodes, which is caused by a nanostructuring effect of the underlying gold during anodization. The stability and biocompatibility of the device are demonstrated by measuring action potentials from cardiomyocyte-like cells growing on top of the chip. Cross sections of the cell-surface interface reveal that the cell membrane seals the nanoporous alumina layer without bending into the sub-50 nm apertures. The nanoporous microelectrode array device may be used as a platform for combining extracellular recording of cell activity with stimulating topographical cues.

Minimal Synthetic Cells to Study Integrin-Mediated Adhesion

To shed light on cell-adhesion-related molecular pathways, synthetic cells offer the unique advantage of a well-controlled model system with reduced molecular complexity. Herein, we show that liposomes with the reconstituted platelet integrin αIIbβ3 as the adhesion-mediating transmembrane protein are a functional minimal cell model for studying cellular adhesion mechanisms in a defined environment. The interaction of these synthetic cells with various extracellular matrix proteins was analyzed using a quartz crystal microbalance with dissipation monitoring. The data indicated that integrin was functionally incorporated into the lipid vesicles, thus enabling integrin-specific adhesion of the engineered liposomes to fibrinogen- and fibronectin-functionalized surfaces. Then, we were able to initiate the detachment of integrin liposomes from these surfaces in the presence of the peptide GRGDSP, a process that is even faster with our newly synthesized peptide mimetic SN529, which specifically inhibits the integrin αIIbβ3.

Model systems for studying cell adhesion and biomimetic actin networks

Summary Many cellular processes, such as migration, proliferation, wound healing and tumor progression are based on cell adhesion. Amongst different cell adhesion molecules, the integrin receptors play a very significant role. Over the past decades the function and signalling of various such integrins have been studied by incorporating the proteins into lipid membranes. These proteolipid structures lay the foundation for the development of artificial cells, which are able to adhere to substrates. To build biomimetic models for studying cell shape and spreading, actin networks can be incorporated into lipid vesicles, too. We here review the mechanisms of integrin-mediated cell adhesion and recent advances in the field of minimal cells towards synthetic adhesion. We focus on reconstituting integrins into lipid structures for mimicking cell adhesion and on the incorporation of actin networks and talin into model cells.

Adhesion and survival of electrogenic cells on gold nanopillar array electrodes

Cell-electrode interfaces play a critical role in extracellular recording. Enlarging the electrode surface area with nanostructures yields higher signal-to-noise-ratios due to lower interface impedance. Adhesion and viability of various cell types on large-scale gold nanopillar electrodes to improve cell-electrode coupling were investigated. Cardiac muscle and human embryonic kidney cells survived and adhered well on gold nanopillars. The muscle cells even protruded into inter-pillar cavities with diameters below 100 nm. However, an unexpectedly low viability and adhesion of primary rat neurons was observed on nanopillars. A cross-sectional analysis of the cell-nanopillar interface showed large distances between neuronal cell bodies and nanopillars, whereas the neurites adhered tightly. Furthermore, actin assembly within the neuronal growth cones was modified on nanopillars. In summary, the adhesion response of the investigated cell lines will be beneficial for improved extracellular signalling, whereas a better understanding of neuronal responses to nanotopographies is required to enhance the neuronal viability.

Nanopore diameters tune strain in extruded fibronectin fibers

Fibronectin is present in the extracellular matrix and can be assembled into nanofibers in vivo by undergoing conformational changes. Here, we present a novel approach to prepare fibronectin nanofibers under physiological conditions using an extrusion approach through nanoporous aluminum oxide membranes. This one-step process can prepare nanofiber bundles up to a millimeter in length and with uniform fiber diameters in the nanometer range. Most importantly, by using different pore diameters and protein concentrations in the extrusion process, we could induce varying lasting structural changes in the fibers, which were monitored by Förster resonance energy transfer and should impose different physiological functions.

Single-molecule mechanics of protein-labelled DNA handles

Summary DNA handles are often used as spacers and linkers in single-molecule experiments to isolate and tether RNAs, proteins, enzymes and ribozymes, amongst other biomolecules, between surface-modified beads for nanomechanical investigations. Custom DNA handles with varying lengths and chemical end-modifications are readily and reliably synthesized en masse, enabling force spectroscopic measurements with well-defined and long-lasting mechanical characteristics under physiological conditions over a large range of applied forces. Although these chemically tagged DNA handles are widely used, their further individual modification with protein receptors is less common and would allow for additional flexibility in grabbing biomolecules for mechanical measurements. In-depth information on reliable protocols for the synthesis of these DNA–protein hybrids and on their mechanical characteristics under varying physiological conditions are lacking in literature. Here, optical tweezers are used to investigate different protein-labelled DNA handles in a microfluidic environment under different physiological conditions. Digoxigenin (DIG)-dsDNA-biotin handles of varying sizes (1000, 3034 and 4056 bp) were conjugated with streptavidin or neutravidin proteins. The DIG-modified ends of these hybrids were bound to surface-modified polystyrene (anti-DIG) beads. Using different physiological buffers, optical force measurements showed consistent mechanical characteristics with long dissociation times. These protein-modified DNA hybrids were also interconnected in situ with other tethered biotinylated DNA molecules. Electron-multiplying CCD (EMCCD) imaging control experiments revealed that quantum dot–streptavidin conjugates at the end of DNA handles remain freely accessible. The experiments presented here demonstrate that handles produced with our protein–DNA labelling procedure are excellent candidates for grasping single molecules exposing tags suitable for molecular recognition in time-critical molecular motor studies.

Force measurements of the disruption of the nascent polypeptide chain from the ribosome by optical tweezers

We show that optical tweezers are a valuable tool to study the co‐translational folding of a nascent polypeptide chain at the ribosome in real‐time. The aim of this study was to demonstrate that a stable and intact population of ribosomes can be tethered to polystyrene beads and that specific hook‐ups to the nascent polypeptide chain by dsDNA handles, immobilized on a second bead, can be detected. A rupture force of the nascent chain in the range of 10–50 pN was measured, which demonstrates that the system is anchored to the surface in a stable and specific way. This will allow in numerous future applications to follow protein folding using much lower forces.

Template-assisted extrusion of biopolymer nanofibers under physiological conditions

Biomedical applications ranging from tissue engineering to drug delivery systems require versatile biomaterials based on the scalable and tunable production of biopolymer nanofibers under physiological conditions. These requirements can be successfully met by a novel extrusion process through nanoporous aluminum oxide templates, which is presented in this study. With this simple method we are able to control the nanofiber diameter by chosing the size of the nanopores and the concentration of the biopolymer feed solution. Nanofiber assembly into different hierarchical fiber arrangements can be achieved with a wide variety of different proteins ranging from the intracellular proteins actin, α-actinin and myosin to the extracellular matrix components collagen, fibronectin, fibrinogen, elastin and laminin. The extrusion of nanofibers can even be applied to the polysaccharides hyaluronan, chitosan and chondroitin sulphate. Moreover, blends of different proteins or proteins and polysaccharides can be extruded into composite nanofibers. With these features our template-assisted extrusion process will lead to new avenues in the development of nanofibrous biomaterials.