Highly Customizable 3D Microelectrode Arrays for In Vitro and In Vivo Neuronal Tissue Recordings.

Planar microelectrode arrays (MEAs) for - in vitro or in vivo - neuronal signal recordings lack the spatial resolution and sufficient signal-to-noise ratio (SNR) required for a detailed understanding of neural network function and synaptic plasticity. To overcome these limitations, a highly customizable three-dimensional (3D) printing process is used in combination with thin film technology and a self-aligned template-assisted electrochemical deposition process to fabricate 3D-printed-based MEAs on stiff or flexible substrates. Devices with design flexibility and physical robustness are shown for recording neural activity in different in vitro and in vivo applications, achieving high-aspect ratio 3D microelectrodes of up to 33:1. Here, MEAs successfully record neural activity in 3D neuronal cultures, retinal explants, and the cortex of living mice, thereby demonstrating the versatility of the 3D MEA while maintaining high-quality neural recordings. Customizable 3D MEAs provide unique opportunities to study neural activity under regular or various pathological conditions, both in vitro and in vivo, and contribute to the development of drug screening and neuromodulation systems that can accurately monitor the activity of large neural networks over time.

Development and in vitro validation of flexible intraretinal probes.

The efforts to improve the treatment efficacy in blind patients with retinal degenerative diseases would greatly benefit from retinal activity feedback, which is lacking in current retinal implants. While the door for a bidirectional communication device that stimulates and records intraretinally has been opened by the recent use of silicon-based penetrating probes, the biological impact induced by the insertion of such rigid devices is still unknown. Here, we developed for the first time, flexible intraretinal probes and validated in vitro the acute biological insertion impact in mouse retinae compared to standard silicon-based probes. Our results show that probes based on flexible materials, such as polyimide and parylene-C, in combination with a narrow shank design 50 µm wide and 7 µm thick, and the use of insertion speeds as high as 187.5 µm/s will successfully penetrate the retina, reduce the footprint of the insertion to roughly 2 times the cross-section of the probe, and induce low dead cell counts, while keeping the vitality of the tissue and recording the neural activity at different depths.

Optimized Continuous Application of Hyperpolarized Xenon to Liquids.

In recent years, NMR with hyperpolarized (HP) xenon inside functionalized host structures (e.g., cryptophanes) have become a potential candidate for the direct observation of metabolic processes (i.e., molecular imaging). A critical issue for real applications is the dissolution of the HP-gas in the liquid which contains the host. In this work, we present recent developments for an improved and controlled dissolution of HP-Xe in liquids using hollow fiber membranes and different compressor systems. The designed apparatus consists of a compressor and a membrane unit. The compressor provides HP-129Xe continuously at small adjustable pressures and in a polarization-preserving way. The membrane unit enables a molecular solution of the HP-gas in aqueous liquids, avoiding the formation of bubbles or even foams. Two different types of compressors were tested in terms of function and useful materials. Special emphasis was put on a systematic reduction of transfer losses in the gas and liquid phase. In order to optimize the system parameters, several physical models were developed to describe the transport and the losses of nuclear polarization. Finally, the successful implementation was demonstrated in several experiments. HP-Xe was dissolved in an aqueous cryptophane-A-(OCH2COOH)6 solution, and stable Xe signals could be measured over 35 min, only limited by the size of the gas reservoir. Such long and stable experimental conditions enabled the study of chemical exchange of xenon between cryptophane and water environments even for a time-consuming 2D NMR experiment. The good signal stability over the measurement time allowed an exact determination of the residence time of the Xe atom inside the cryptophane, resulting in an average residence time of 44.5 ± 2.7 ms.

Facile, non-destructive characterization of 2d photonic crystals using UV-vis-spectroscopy.

We present a simple and non-destructive method for characterizing and quantifying the quality of two-dimensional (2D) close-packed arrays of submicron dielectric spheres. Utilizing radiative losses of photonic modes created by the 2D crystals into dielectric substrates we are able to monitor the quality of the particle monolayer during assembly and the size evolution of the individual particles during dry etching. Using an advanced interfacial assembly technique we prepare particle monolayers on glass and characterize the spectral behaviour of the radiative loss regarding different lattice constants, dielectric substrates and layer qualities. The effect of diameter reduction during dry etching is analysed and a simple model is proposed, which enables non-destructive, on spot characterization of the particle layer with sub-20 nm resolution using UV-vis spectroscopy.

Shell-binary nanoparticle materials with variable electrical and electro-mechanical properties.

Nanoparticle (NP) materials with the capability to adjust their electrical and electro-mechanical properties facilitate applications in strain sensing technology. Traditional NP materials based on single component NPs lack a systematic and effective means of tuning their electrical and electro-mechanical properties. Here, we report on a new type of shell-binary NP material fabricated by self-assembly with either homogeneous or heterogeneous arrangements of NPs. Variable electrical and electro-mechanical properties were obtained for both materials. We show that the electrical and electro-mechanical properties of these shell-binary NP materials are highly tunable and strongly affected by the NP species as well as their corresponding volume fraction ratio. The conductivity and the gauge factor of these shell-binary NP materials can be altered by about five and two orders of magnitude, respectively. These shell-binary NP materials with different arrangements of NPs also demonstrate different volume fraction dependent electro-mechanical properties. The shell-binary NP materials with a heterogeneous arrangement of NPs exhibit a peaking of the sensitivity at medium mixing ratios, which arises from the aggregation induced local strain enhancement. Studies on the electron transport regimes and micro-morphologies of these shell-binary NP materials revealed the different mechanisms accounting for the variable electrical and electro-mechanical properties. A model based on effective medium theory is used to describe the electrical and electro-mechanical properties of such shell-binary nanomaterials and shows an excellent match with experiment data. These shell-binary NP materials possess great potential applications in high-performance strain sensing technology due to their variable electrical and electro-mechanical properties.

N-Cadherin modified lipid bilayers promote neural network formation and circuitry.

Neural adhesion, maturation, and the correct wiring of the brain to establish each neuron's intended connectivity are controlled by complex interactions of bioactive molecules such as ligands, growth factors, or enzymes. The correct pairing of adjacent neurons is thought to be highly regulated by ligand-mediated cell-cell adhesion proteins, which are known to induce signaling activities. We developed a new platform consisting of supported lipid bilayers incorporated with Fc-chimera synaptic proteins like ephrinA5 or N-cadherin. We extensively characterized their function employing a quartz crystal microbalance with dissipation (QCM-D), calcium imaging, and immunofluorescence analysis. Our biomimetic platform has been shown to promote neural cell adhesion and to improve neural maturation at day in vitro 7 (DIV7) as indicated by an elevated expression of synaptophysin.

Observation of chemically protected polydimethylsiloxane: towards crack-free PDMS.

The current modification of polydimethylsiloxane (PDMS) substrates via oxygen plasma treatment causes surface cracks. Here, we demonstrate a method to prevent crack formation by chemical treatment. Chemical modification renders the surface hydrophilic for several days and is effective in preserving the elasticity of the PDMS surface at the nanoscale level.

Temperature-Dependent Electron Transport in Single Terphenyldithiol Molecules.

Analyzing the electronic properties of individual terphenyldithiol (TPT) molecules in a temperature range of 30-300 K using cryogenic mechanically controllable break junctions, we observe an unexpected change of the transport mechanism with temperature for this linear and symmetric aromatic molecule. Whereas the work function (∼3.8 eV) and molecular energy level (∼0.8 to ∼1 eV depending on the analysis of the data) of TPT are temperature-independent, elastic tunneling dominates charge transport at low temperatures, whereby an inelastic transport (via hopping) sets in at about 100 K. The molecular level of TPT lies around 1 eV and is temperature-independent. This unusual temperature dependence agrees with recent predictions for other short molecules using density-functional-based transport studies as well as experimental observations obtained for similar relatively short rodlike molecules.

Surface coupling strength of gold nanoparticles affects cytotoxicity towards neurons.

Gold nanoparticles (AuNPs) are versatile nanomaterials which are frequently used to manipulate and study cellular behavior on the nanometer scale. However, it has been recognized that freely diffusing colloidal particles can possess severe cytotoxicity. One strategy to overcome this harmful side effect is to tether the nanoparticles to sample surfaces. In this study, the cytotoxicity of immobilized AuNPs is investigated as a function of their sizes, surface density, and binding strength. The AuNPs are modified with positively charged aminoalkyl thiol molecules to promote the cell adhesion, while their background is passivated. Primary cortical neurons are cultured on the particle modified samples and the survival of the cells is investigated. This study reveals that besides the particle size, the particle binding strength influences the cytotoxicity during long time culture. Weakly bound particles dramatically decrease the survival of neurons while strongly bound particles hardly harm the neurons. More importantly, it is found that weakly bound AuNPs can cause higher cytotoxic effects on cells than colloidal particles dispersed in the culture medium. These results propose that the toxic effect of surface bound particles can be severe and therefore requires consideration, if nanoparticle modified surfaces are used for cell experiments.

Single Molecule Characterization of UV-Activated Antibodies on Gold by Atomic Force Microscopy.

The interaction between proteins and solid surfaces can influence their conformation and therefore also their activity and affinity. These interactions are highly specific for the respective combination of proteins and solids. Consequently, it is desirable to investigate the conformation of proteins on technical surfaces, ideally at single molecule level, and to correlate the results with their activity. This is in particular true for biosensors where the conformation-dependent target affinity of an immobilized receptor determines the sensitivity of the sensor. Here, we investigate for the first time the immobilization and orientation of antibodies (Abs) photoactivated by a photonic immobilization technique (PIT), which has previously demonstrated to enhance binding capabilities of antibody receptors. The photoactivated immunoglobulins are immobilized on ultrasmooth template stripped gold films and investigated by atomic force microscopy (AFM) at the level of individual molecules. The observed protein orientations are compared with results of nonactivated antibodies adsorbed on similar gold films and mica reference samples. We find that the behavior of Abs is similar for mica and gold when the protein are not treated (physisorption), whereas smaller contact area and larger heights are measured when Abs are treated (PIT). This is explained by assuming that the activated antibodies tend to be more upright compared with nonirradiated ones, thereby providing a better exposure of the binding sites. This finding matches the observed enhancement of Abs binding efficiency when PIT is used to functionalize gold surface of QCM-based biosensors.

HP-Xe to go: Storage and transportation of hyperpolarized (129)Xenon.

Recently the spin-lattice relaxation time T1 of hyperpolarized (HP)-(129)Xe was significantly improved by using uncoated and Rb-free storage vessels of GE180 glass. For these cells, a simple procedure was established to obtain reproducible wall relaxation times of about 18 h. Then the limiting relaxation mechanism in pure Xe is due to the coupling between the nuclear spins and the angular momentum of the Xe-Xe van-der-Waals-molecules. This mechanism can be significantly reduced by using different buffer gases of which CO2 was discovered to be the most efficient so far. From these values, it was estimated that for a 1:1 mixture of HP-Xe with CO2 a longitudinal relaxation time of about 7 h can be expected, sufficient to transport HP-Xe from a production to a remote application site. This prediction was verified for such a mixture at a total pressure of about 1 bar in a 10 cm glass cell showing a storage time of T1≈9 h (for T1(wall)=(34±9) h) which was transported inside a magnetic box over a distance of about 200 km by car.

Reconstitution of Fusion Proteins in Supported Lipid Bilayers for the Study of Cell Surface Receptor-Ligand Interactions in Cell-Cell Contact.

Bioactive molecules such as adhesion ligands, growth factors, or enzymes play an important role in modulating cell behavior such as cell adhesion, spreading, and differentiation. Deciphering the mechanism of ligand-mediated cell adhesion and associated signaling is of great interest not only for fundamental biophysical investigations but also for applications in medicine and biotechnology. In the presented work, we developed a new biomimetic platform that enables culturing primary neurons and testing cell surface-receptor ligand interactions in cell-cell contacts as, e.g., in neuronal synapses. This platform consists of a supported lipid bilayer modified with incorporated neuronal adhesion proteins conjugated with the Fc-domain of IgG (ephrin A5 Fc-chimera). We extensively characterized properties of these protein containing bilayers using fluorescence recovery after photobleaching (FRAP), quartz crystal microbalance with dissipation (QCM-D), and immunostaining. We conclude that the Fc-domain is the part responsible for the incorporation of the protein into the bilayer. The biomimetic platform prepared by this new approach was able to promote neuronal cell adhesion and maintain growth as well as facilitate neuronal maturation as shown by electrophysiological measurements. We believe that our approach can be extended to insert other proteins to create a general culture platform for neurons and other cell types.

Ultra-thin resin embedding method for scanning electron microscopy of individual cells on high and low aspect ratio 3D nanostructures.

The preparation of biological cells for either scanning or transmission electron microscopy requires a complex process of fixation, dehydration and drying. Critical point drying is commonly used for samples investigated with a scanning electron beam, whereas resin-infiltration is typically used for transmission electron microscopy. Critical point drying may cause cracks at the cellular surface and a sponge-like morphology of nondistinguishable intracellular compartments. Resin-infiltrated biological samples result in a solid block of resin, which can be further processed by mechanical sectioning, however that does not allow a top view examination of small cell-cell and cell-surface contacts. Here, we propose a method for removing resin excess on biological samples before effective polymerization. In this way the cells result to be embedded in an ultra-thin layer of epoxy resin. This novel method highlights in contrast to standard methods the imaging of individual cells not only on nanostructured planar surfaces but also on topologically challenging substrates with high aspect ratio three-dimensional features by scanning electron microscopy.

Inkjet printing of UV-curable adhesive and dielectric inks for microfluidic devices.

Bonding of polymer-based microfluidics to polymer substrates still poses a challenge for Lab-On-a-Chip applications. Especially, when sensing elements are incorporated, patterned deposition of adhesives with curing at ambient conditions is required. Here, we demonstrate a fabrication method for fully printed microfluidic systems with sensing elements using inkjet and stereolithographic 3D-printing.

In situ fabrication of ultrathin porous alumina and its application for nanopatterning Au nanocrystals on the surface of ion-sensitive field-effect transistors.

In situ fabrication in a single step of thin films of alumina exhibiting a thickness of less than 100 nm and nanopores with a highly regular diameter distribution in order to pattern nanostructures over field-effect devices is a critical issue and has not previously been demonstrated. Here we report the fabrication in situ of 50 nm thick ultrathin nanoporous alumina membranes with a regular pore size directly over metal-free gate ion-sensitive field-effect transistors. Depositing thin films of aluminum by an electron beam at a relatively low rate of deposition on top of chips containing the transistors and using a conventional single-step anodization process permits the production of a well-adhering nanoporous ultrathin layer of alumina on the surface of the devices. The anodization process does not substantially affect the electrical properties of the transistors. The small thickness and pore size of ultrathin alumina membranes allow them to be sequentially employed as masks for patterning Au nanocrystals grown by an electroless approach directly on the top of the transistors. The patterning process using a wet chemical approach enables the size of the patterned crystals to be controlled not only by the dimensions of the pores of alumina, but also by the concentration of the reactants employed. Surface modification of these nanocrystals with alkanethiol molecules demonstrates that the electrostatic charge of the functional groups of the molecules can modulate the electrical characteristics of the transistors. These results represent substantial progress towards the development of novel nanostructured arrays on top of field-effect devices that can be applied for chemical sensing or non-volatile memories.

Patterned self-assembly of gold nanoparticles on chemical templates fabricated by soft UV nanoimprint lithography.

Chemical templates for the patterned immobilization of gold nanoparticles were fabricated by soft UV nanoimprint lithography. The template structures were fabricated by means of the consecutively performed process steps of nanoimprint lithography, reactive ion etching, chemical functionalization with amino groups, and lift-off of imprint resist. These chemical templates were used for the defined assembly of 20 nm diameter citrate stabilized gold nanoparticles from aqueous solution. By reducing the ionic strength of the solution, one- and zero-dimensional particle assemblies were generated on sub-100-nm template structures. By this means, the pattern resolution predefined by the lithography process could be easily enhanced by dilution of the nanoparticle solution.

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.

Extracellular recording of glycine receptor chloride channel activity as a prototype for biohybrid sensors.

There is a continuously growing scientific and technological interest to develop and improve the application of artificial sensors. Biological components which are capable to transduce neutral signals into specific, robust and reproducible indicators frame an attractive alternative to construct biohybrid sensors. Since naturally "occurring" biosensors are only sparsely compatible with artificial devices, genetic engineering of eukaryotic cells provides an attractive approach, where cells can be tailored such to detect target compounds with exquisite specificity and sensitivity. We have developed the prototype for a single-cell-based anion-selective biohybrid sensor. HEK293 cells were stably transfected with a gene encoding glycine receptor alpha(1) subunits. These cells were employed as transducers for glycine-evoked chloride currents in a concentration-dependent way. Cultured on substrate-integrated micro-devices, anionic membrane currents of cells were monitored extracellularly with field-effect transistors (FETs) and gold microelectrode arrays (MEAs). The results supported predictions of state-of-the-art models for cell-sensor coupling mechanisms and confirmed that extracellularly recorded anion currents cause similar signals, regardless whether obtained with field-effect transistors or microelectrodes. The whole-cell sensor successfully tracked glycine concentrations differing by three orders of magnitude. To our knowledge this contribution for the first time marks the functional characterization of an anion-selective biohybrid sensor.

Field-effect devices for detecting cellular signals.

The integration of living cells together with silicon field-effect devices challenges a new generation of biosensors and bioelectronic devices. Cells are representing highly organised complex systems, optimised by millions of years of evolution and offering a broad spectrum of bioanalytical receptor "tools" such as enzymes, nucleic acids proteins, etc. Their combination with semiconductor-based electronic chips allows the construction of functional hybrid systems with unique functional and electronic properties for both fundamental studies and biosensoric applications. This review article summarises recent advances and trends in research and development of cell/transistor hybrids (cell-based field-effect transistors) as well as light-addressable potentiometric sensors.

The use of microelectrode array (MEA) to study the protective effects of potassium channel openers on metabolically compromised HL-1 cardiomyocytes.

The microelectrode array (MEA) was used to evaluate the cardioprotective effects of adenosine triphosphate sensitive potassium (K(ATP)) channel activation using potassium channel openers (KCOs) on HL-1 cardiomyocytes subjected to acute chemically induced metabolic inhibition. Beat frequency and extracellular action potential (exAP) amplitude were measured in the presence of metabolic inhibitors (sodium azide (NaN(3)) or 2-deoxyglucose (2-DG)) or KCOs (pinacidil (PIN, a cyanoguanidine derivative, activates sarcolemmal K(ATP) channels) or SDZ PCO400 (SDZ, a benzopyran derivative, activates mitochondrial K(ATP) channels)). The protective effects of these KCOs on metabolically inhibited HL-1 cells were subsequently investigated. Signal shapes indicated that NaN(3) and 2-DG reduced the rate of the sodium (Na(+)) influx signal as reflected by a reduction in beat frequency. PIN and SDZ appeared to reduce both rate of depolarization and extent of the Na(+) influx signals. Pre-treating cardiomyocytes with PIN (0.1 mM), but not SDZ, prevented the reduction of beat frequency associated with NaN(3)- or 2-DG-induced metabolic inhibition. The exAP amplitude was not affected by either KCO. The cardioprotective effect of PIN relative to SDZ may be due to the opening of different K(ATP) channels. This metabolic inhibition model on the MEA may provide a stable platform for the study of cardiac pathophysiology in the future.