Andreas Offenhäusser

Field-effect transistor array for monitoring electrical activity from mammalian neurons in culture

A field-effect transistor (FET) array has been fabricated and used for recording of electrical signals from neural cells. The array consists of p-channel FETs with non-metalized gates. The size of the gates of the 16 FETs are from 28 x 12 microns2 down to 10 x 4 microns2 and are arranged in a 4 x 4 matrix on 200 microns centers. For the device fabrication process we have especially focused on high sensitivity, good long-term stability in physiological conditions, and sufficient reduced signal-to-noise ratio. Special care was taken on the encapsulation technique of the device to allow surface modification based on the self-assembly technique. It can be shown that the microelectronic device surface can be modified with a synthetic peptide linked to the surface. Tailoring of the surface composition using this method allows hippocampal neurons to adhere and grow for days. More importantly, these cells develop typical electrical characteristics when cultured on this artificial surface. Using this approach neuron-FET couplings were recorded.

Ordered networks of rat hippocampal neurons attached to silicon oxide surfaces.

The control of neuronal cell position and outgrowth is of fundamental interest in the development of applications ranging from cellular biosensors to tissue engineering. We have produced rectangular networks of functional rat hippocampal neurons on silicon oxide surfaces. Attachment and network formation of neurons was guided by a geometrical grid pattern of the adhesion peptide PA22-2 which matches in sequence a part of the A-chain of laminin. PA22-2 was applied by contact printing onto the functionalised silicon oxide surface and was immobilised by hetero-bifunctional cross-linking with sulfo-GMBS. Geometric pattern matching was achieved by microcontact printing using a polydimethylsiloxane (PDMS) stamp. In this way the produced grid pattern ranged from 3 to 20 microm in line width and from 50 to 100 microm in line distances. As shown by atomic force microscopy (AFM), line widths and line distances of the peptide pattern differ less than 0.5 microm from the used PDMS stamp. The height of the layer of immobilised PA22-2 was approximately 3.5 nm implying the layer to be monomolecular. Immobilised PA22-2 was capable of binding anti-PA22-2 antibodies indicating that the function of the peptide was not compromised by immobilisation. Rat hippocampal neurons, cultured at low density in serum-free medium, were applied to the growth matrix of PA22-2-coated substrates and, within 1-3 h of culture, formed a network-like pattern that more or less matched the printed grid. Reliability and reproducibility of neuronal network formation depended on the geometry, line width and node diameter of the grid pattern. The immobilised neurons showed resting membrane potentials comparable with controls and, already after 1 day of culture, were capable of eliciting action potentials. The suitability of the immobilised neurons for the study of man-made neural networks and for multi-site recordings from a functional neuronal network is discussed.

Graphene Transistor Arrays for Recording Action Potentials from Electrogenic Cells

The development of the future generation of neuroprosthetic devices will require the advancement of novel solid-state sensors and actuators with a further improvement in the signal detection capability, a superior stability in biological environments, and a more suitable compatibility with living tissue. To date, interfacing of living cells and tissue with solid-state electronic devices has mainly been based on conventional silicon technology, in particular using Si metal-oxide-semiconductor fi eld-effect transistor (MOSFET) structures. [ 1 ] However, some of the drawbacks associated with this technology, such as its limited stability in aqueous environments [ 2 ] and a relatively high electrical noise, [ 3 ] have triggered the study of alternative materials and technologies. [ 4–11 ] In this respect, solution-gated fi eldeffect transistors (SGFETs) based on Si-nanowires, [ 4 ] AlGaN/ GaN heterostructures, [ 5 ] H-terminated diamond, [ 6 , 7 ] carbon nanotubes, [ 8 ] and, more recently, graphene [ 9–11 ] have been investigated as sensing devices. Among these materials, graphene is a particularly attractive candidate for bioelectronic applications, due to its remarkable physical and chemical properties. The extremely high charge carrier mobility in graphene [ 12 ] leads to a fi eld-effect transistor (FET) performance that is superior to most known semiconductors. [ 13 ] In addition, graphene is known to possess good chemical stability [ 14 ] and biocompatibility, [ 15 ] which is crucial not only for integration with biological systems, but also for the operation of fi eld-effect devices without a protective dielectric layer. Furthermore, the facile integration of graphene electronics with fl exible substrates paves the way for the development of fl exible devices, an important requirement for the design of biomedical implants with reduced tissue damage and scarring. [ 16 ] The use of graphene-based solutiongated fi eld-effect transistors (G-SGFETs) for the detection of cell signals has already been demonstrated on a fundamental level by using a single transistor on exfoliated graphene. [ 8 ] Despite this promising preliminary result, a successful graphene-based technology for the applications envisioned above requires the demonstration of arrays of graphene transistors, which can

The peptide-tethered lipid membrane as a biomimetic system to incorporate cytochrome c oxidase in a functionally active form

Abstract Peptide-supported lipid bilayers are investigated as a new class of solidsupported membranes tethered to the support by a peptide spacer. They are referred to as peptide tethered lipid membranes (tBLMs), formed by the fusion of liposomes with a thiopeptide-lipid monolayer chemisorbed on a gold support. Peptide tBLMs are designed as a biomimetic system to investigate integral membrane proteins. As an example, cytochrome c oxidase (COX) from bovine heart is incorporated into the preformed peptide tBLM by dilution of the solubilised protein below the critical micellar concentration. The formation of the lipid film as well as the incorporation of the protein were monitored by surface plasmon resonance spectroscopy and surface plasmon fluorescence spectroscopy. COX is activated by adding the reduced form of cytochrome c to the air-saturated buffer solution. Using electrochemical techniques, such as square wave voltammetry (SWV) and chronoamperometry (CA), the direct electron transfer between COX and the gold electrode is observed as well as proton transport from the inside to the outside across the lipid bilayer. Proton transport is then further investigated using impedance spectroscopy, although the electrode is shown to be only partially (70%) covered with a bilayer while defect domains with only a monolayer of peptide or peptide-lipid coexist (approx. 30%). Proton transport carried out by the COX is shown to be voltage dependent. This transport is indicated as a resistance in parallel to the resistance of the lipid film. As a consequence, the total resistance decreases as a function of the concentration of cytochrome c and increases again either by removal of the substrate or by addition of cyanide as an inhibitor of COX. The conductance in the presence of the activated enzyme correlates with the known turnover rate of COX. These experiments demonstrate the possibility to assess the activity of integral membrane proteins incorporated in peptide tBLMs using electrochemical techniques. The system could thus be promising for screening as well as biosensor applications.

Recording of cell action potentials with AlGaN∕GaN field-effect transistors

An AlGaN∕GaN electrolyte gate field-effect transistor array for the detection of electrical cell signals has been realized. The low-frequency noise power spectral density of these devices exhibits a 1∕f characteristic with a dimensionless Hooge parameter of 5×10−3. The equivalent gate-input noise under operation conditions has a peak-to-peak amplitude of 15μV, one order of magnitude smaller than for common silicon-based devices used for extracellular recordings. Extracellular action potentials from a confluent layer of rat heart muscle cells cultivated directly on the nonmetallized gate surface were recorded with a signal amplitude of 75μV and a signal-to-noise ratio of 5:1.

Cell-Transistor Hybrid Systems and Their Potential Applications

Electrogenic cells fire spontaneous or triggered action potentials (transient changes of their membrane potentials) and can be electronically coupled to external electrodes (arrays). Signals from rat heart-muscle cells were recorded by a field-effect transistor and the results described on the basis of an equivalent circuit. This technique has potential applications in drug screening, such as measuring the dose-response curve of isoproterenol, a beta-adrenergic agonist with a positive chronotropic effect.

PDMS device for patterned application of microfluids to neuronal cells arranged by microcontact printing.

A microfluidic device in polydimethylsiloxane (PDMS) consisting of an eight lines micro-injection array integrated in a base flow channel has been realized. The device is assembled from multiple PDMS parts, which have been moulded using notably micromachined masters in SU-8 photoresist. In contact with a planar substrate, up to eight independent laminar flow lines with cross-sections of 100 x 200 microm(2) can be generated. Dedicated for the application of pharmaceutical compounds to electrogenic cells in vitro, this device was tested with a neuronal cell line, Mz1-cells. These were cultured on lines of laminin deposited onto polystyrene substrates by microcontact printing. We were able to inject into this culture multiple lines of coloured PBS in parallel to the orientation of cellular growth. No mixing between the individual flow lines did occur.

Cardiomyocyte-transistor-hybrids for sensor application

An extracellular recording system has been designed for the detection of electrical cell signals using p-channel or n-channel field-effect transistor (FET) arrays with non-metallized gates. Signals from rat heart muscle cell were recorded by these devices and the results described on the basis of an equivalent circuit. This technique is sensitive enough to detect minute changes of the extracellular membrane voltage and has potential applications in drug screening. We show that known cardiac stimulants (isoproterenol, norepinephrine) and relaxants (verapamil, carbamylcholine) have characteristic effects on the heart cells in terms of the changes of beat frequencies in the absence or presence of corresponding agents.

Proton transport through a peptide-tethered bilayer lipid membrane by the H+-ATP synthase from chloroplasts measured by impedance spectroscopy

A lipid membrane was tethered to a gold film by a peptide spacer molecule terminated by a sulfhydryl group. Membranes were formed by fusion of liposomes prepared from egg phosphatidylcholine on self assembled monolayers of the thiolipopeptide Myr-Lys(Myr)-Ser-Ser-Pro-Ala-Ser-Ser-Ala-Ala-Ser-Ala-Cys-amide mixed with mercaptoethanol as a diluent molecule or lateral spacer. These mixed films, although not representing a perfect lipid bilayer, have been shown to retain the activity of incorporated H(+)-ATP synthases from chloroplasts in contrast to films prepared from the pure thiolipopeptide. The activity of the protein was demonstrated by impedance spectroscopy. The resistance decreased due to proton transport across the lipid film, which occurs as a consequence of adenosine triphosphate (ATP) hydrolysis. Several effects previously determined from kinetic measurements of the enzyme reconstituted in liposomes such as saturation with respect to the substrate (ATP), inhibition by venturicidin, activation by a positive potential pulse and increase of the proton current as a function of increasingly negative potentials have been confirmed also for this tethered membrane system. Changes in the impedance spectra due to the addition of ATP were fully reversible.

Extended gate electrode arrays for extracellular signal recordings

Abstract We have fabricated arrays of planar gold electrodes arranged in a matrix of 8×8 with active areas ranging from 6 to 30 μm in diameter. An electronic amplification circuitry based on commercial junction field-effect transistors was used where the gold sensor fields act as extended gate electrodes (EGE) of the transistors, which leads to a new approach for long-term extracellular recording systems in vitro. The high input resistance of the amplification circuitry allows the use of small planar bare gold electrodes without further modification which therefore extends the frequency range of the measuring set-up down to the DC-level. The performance of our recording system has been tested using rat cardiac myocytes cultured directly on the device surface. The recorded signals were then compared in shape and size with recordings performed with a similar extracellular measurement set-up based directly on field-effect transistors with non-metallized gate electrodes. By simulations we could show the influence of the electrode capacitance on the time-course and on the size of the measured signals.