Planar patch clamp for neuronal networks--considerations and future perspectives.

The patch-clamp technique is generally accepted as the gold standard for studying ion channel activity allowing investigators to either "clamp" membrane voltage and directly measure transmembrane currents through ion channels, or to passively monitor spontaneously occurring intracellular voltage oscillations. However, this resulting high information content comes at a price. The technique is labor-intensive and requires highly trained personnel and expensive equipment. This seriously limits its application as an interrogation tool for drug development. Patch-clamp chips have been developed in the last decade to overcome the tedious manipulations associated with the use of glass pipettes in conventional patch-clamp experiments. In this chapter, we describe some of the main materials and fabrication protocols that have been developed to date for the production of patch-clamp chips. We also present the concept of a patch-clamp chip array providing high resolution patch-clamp recordings from individual cells at multiple sites in a network of communicating neurons. On this chip, the neurons are aligned with the aperture-probes using chemical patterning. In the discussion we review the potential use of this technology for pharmaceutical assays, neuronal physiology and synaptic plasticity studies.

Priming and testing silicon patch-clamp neurochips.

We report on the systematic and automated priming and testing of silicon planar patch-clamp chips after their assembly in Plexiglas packages and sterilization in an air plasma reactor. We find that almost 90% of the chips are successfully primed by our automated setup, and have a shunt capacitance of between 10 pF and 30 pF. Blocked chips are mostly due to glue invasion in the well, and variability in the manual assembly process is responsible for the distribution in shunt capacitance value. Priming and testing time with our automated setup is less than 5 min per chip, which is compatible with the production of large series for use in electrophysiology experiments.

Polymer peel-off mask for high-resolution surface derivatization, neuron placement and guidance.

We present a dry lift-off method using a chemically resistant spin-on plastic, polyimide, to pattern surfaces with high accuracy and resolution. Using well-known lithographic and reactive ion etching techniques, the spin-on polymer is patterned over a silicon dioxide surface. The plastic efficiently adheres to the silicon dioxide surface during the chemical modification and is readily lifted-off following the derivatization process, permitting highly reliable surface derivatization. The verticality of the reactive ion etch enables sub-micrometer features to be patterned, down to 0.8 µm. The technique is used to pattern neurons on silicon dioxide surfaces: efficient neuron placement over a 4 mm area is shown for patterns larger than 50 µm while process guidance is shown for 10 µm patterns.

Culturing and electrophysiology of cells on NRCC patch-clamp chips.

Due to its exquisite sensitivity and the ability to monitor and control individual cells at the level of ion channels, patch-clamping is the gold standard of electrophysiology applied to disease models and pharmaceutical screens alike. The method traditionally involves gently contacting a cell with a glass pipette filled by a physiological solution in order to isolate a patch of the membrane under its apex. An electrode inserted in the pipette captures ion-channel activity within the membrane patch or, when ruptured, for the whole cell. In the last decade, patch-clamp chips have been proposed as an alternative: a suspended film separates the physiological medium from the culture medium, and an aperture microfabricated in the film replaces the apex of the pipette. Patch-clamp chips have been integrated in automated systems and commercialized for high-throughput screening. To increase throughput, they include the fluidic delivery of cells from suspension, their positioning on the aperture by suction, and automated routines to detect cell-to-probe seals and enter into whole cell mode. We have reported on the fabrication of a silicon patch-clamp chip with optimized impedance and orifice shape that permits the high-quality recording of action potentials in cultured snail neurons; recently, we have also reported progress towards interrogating mammalian neurons. Our patch-clamp chips are fabricated at the Canadian Photonics Fabrication Centre, a commercial foundry, and are available in large series. We are eager to engage in collaborations with electrophysiologists to validate the use of the NRCC technology in different models. The chips are used according to the general scheme represented in Figure 1: the silicon chip is at the bottom of a Plexiglas culture vial and the back of the aperture is connected to a subterranean channel fitted with tubes at either end of the package. Cells are cultured in the vial and the cell on top of the probe is monitored by a measuring electrode inserted in the channel .The two outside fluidic ports facilitate solution exchange with minimal disturbance to the cell; this is an advantage compared to glass pipettes for intracellular perfusion.

From understanding cellular function to novel drug discovery: the role of planar patch-clamp array chip technology.

All excitable cell functions rely upon ion channels that are embedded in their plasma membrane. Perturbations of ion channel structure or function result in pathologies ranging from cardiac dysfunction to neurodegenerative disorders. Consequently, to understand the functions of excitable cells and to remedy their pathophysiology, it is important to understand the ion channel functions under various experimental conditions - including exposure to novel drug targets. Glass pipette patch-clamp is the state of the art technique to monitor the intrinsic and synaptic properties of neurons. However, this technique is labor intensive and has low data throughput. Planar patch-clamp chips, integrated into automated systems, offer high throughputs but are limited to isolated cells from suspensions, thus limiting their use in modeling physiological function. These chips are therefore not most suitable for studies involving neuronal communication. Multielectrode arrays (MEAs), in contrast, have the ability to monitor network activity by measuring local field potentials from multiple extracellular sites, but specific ion channel activity is challenging to extract from these multiplexed signals. Here we describe a novel planar patch-clamp chip technology that enables the simultaneous high-resolution electrophysiological interrogation of individual neurons at multiple sites in synaptically connected neuronal networks, thereby combining the advantages of MEA and patch-clamp techniques. Each neuron can be probed through an aperture that connects to a dedicated subterranean microfluidic channel. Neurons growing in networks are aligned to the apertures by physisorbed or chemisorbed chemical cues. In this review, we describe the design and fabrication process of these chips, approaches to chemical patterning for cell placement, and present physiological data from cultured neuronal cells.

Recordings of cultured neurons and synaptic activity using patch-clamp chips.

Planar patch-clamp chip technology has been developed to enhance the assessment of novel compounds for therapeutic efficacy and safety. However, this technology has been limited to recording ion channels expressed in isolated suspended cells, making the study of ion channel function in synaptic transmission impractical. Recently, we developed single- and dual-recording site planar patch-clamp chips and demonstrated their capacity to record ion channel activity from neurons established in culture. Such capacity provides the opportunity to record from synaptically connected neurons cultured on-chip. In this study we reconstructed, on-chip, a simple synaptic circuit between cultured pre-synaptic visceral dorsal 4 neurons and post-synaptic left pedal dorsal 1 neurons isolated from the mollusk Lymnaea stagnalis. Here we report the first planar patch-clamp chip recordings of synaptic phenomena from these paired neurons and pharmacologically demonstrate the cholinergic nature of this synapse. We also report simultaneous dual-site recordings from paired neurons, and demonstrate dedicated cytoplasmic perfusion of individual neurons via on-chip subterranean microfluidics. This is the first application of planar patch-clamp technology to examine synaptic communication.

Cell to aperture interaction in patch-clamp chips visualized by fluorescence microscopy and focused-ion beam sections.

Patch-clamp is an important method to monitor the electrophysiological activity of cells and the role of pharmacological compounds on specific ion channel proteins. In recent years, planar patch-clamp chips have been developed as a higher throughput approach to the established glass-pipette method. However, proper conditions to optimize the high resistance cell-to-probe seals required to measure the small currents resulting from ion channel activity are still the subject of conjecture. Here, we report on the design of multiple-aperture (sieve) chips to rapidly facilitate assessment of cell-to-aperture interactions in statistically significant numbers. We propose a method to pre-screen the quality of seals based on a dye loading protocol through apertures in the chip and subsequent evaluation with fluorescence confocal microscopy. We also show the first scanning electron micrograph of a focused ion beam section of a cell in a patch-clamp chip aperture.

High-fidelity patch-clamp recordings from neurons cultured on a polymer microchip.

We present a polymer microchip capable of monitoring neuronal activity with a fidelity never before obtained on a planar patch-clamp device. Cardio-respiratory neurons Left Pedal Dorsal 1 (LPeD1) from mollusc Lymnaea were cultured on the microchip's polyimide surface for 2 to 4 hours. Cultured neurons formed high resistance seals (gigaseals) between the cell membrane and the surface surrounding apertures etched in the polyimide. Gigaseal formation was observed without applying external force, such as suction, on neurons. The formation of gigaseals, as well as the low access resistance and shunt capacitance values of the polymer microchip resulted in high-fidelity recordings. On-chip culture of neurons permitted, for the first time on a polymeric patch-clamp device, the recording of high fidelity physiological action potentials. Microfabrication of the hybrid poly(dimethylsiloxane)-polyimide (PDMS-PI) microchip is discussed, including a two-layer PDMS processing technique resulting in minimized shrinking variations.

A novel silicon patch-clamp chip permits high-fidelity recording of ion channel activity from functionally defined neurons.

We report on a simple and high-yield manufacturing process for silicon planar patch-clamp chips, which allow low capacitance and series resistance from individually identified cultured neurons. Apertures are etched in a high-quality silicon nitride film on a silicon wafer; wells are opened on the backside of the wafer by wet etching and passivated by a thick deposited silicon dioxide film to reduce the capacitance of the chip and to facilitate the formation of a high-impedance cell to aperture seal. The chip surface is suitable for culture of neurons over a small orifice in the substrate with minimal leak current. Collectively, these features enable high-fidelity electrophysiological recording of transmembrane currents resulting from ion channel activity in cultured neurons. Using cultured Lymnaea neurons we demonstrate whole-cell current recordings obtained from a voltage-clamp stimulation protocol, and in current-clamp mode we report action potentials stimulated by membrane depolarization steps. Despite the relatively large size of these neurons, good temporal and spatial control of cell membrane voltage was evident. To our knowledge this is the first report of recording of ion channel activity and action potentials from neurons cultured directly on a planar patch-clamp chip. This interrogation platform has enormous potential as a novel tool to readily provide high-information content during pharmaceutical assays to investigate in vitro models of disease, as well as neuronal physiology and synaptic plasticity.

Cell placement and guidance on substrates for neurochip interfaces.

Interface devices such as integrated planar patch-clamp chips are being developed to study the electrophysiological activity of neuronal networks grown in vitro. The utility of such devices will be dependent upon the ability to align neurons with interface features on the chip by controlling neuronal placement and by guiding cell connectivity. In this paper, we present a strategy to accomplish this goal. Patterned chemical modification of SiN surfaces with poly-d-lysine transferred from PDMS stamps was used to promote adhesion and guidance of cryo-preserved primary rat cortical neurons. We demonstrate that these neurons can be positioned and grown over microhole features which will ultimately serve as patch-clamp interfaces on the chip.

Design of high-contrast OLEDs with microcavity effect.

There is a large demand for Organic Light-Emitting Displays (OLEDs) with higher contrast, particularly for outdoor applications. We show that lowering the reflectance of OLEDs, which is required for increasing the contrast, can also lead to a reduction of their efficiency when a small microcavity effect is not maintained in their structure. We describe in details the design of high-contrast bottom-emitting OLEDs that have low reflectance but still maintain a small cavity effect for efficient emission.

High-contrast organic light emitting diodes with a partially absorbing anode.

To be legible in high-ambient light conditions, organic light-emitting-diode displays should be optically designed to have a minimal reflectance without significantly affecting their overall efficiency. We demonstrate the use of an anode consisting of a partially absorbing metal layer and a multilayer distributed Bragg reflector to simultaneously absorb rather than reflect incoming light and to take advantage of a weak microcavity effect in the diode to improve light outcoupling.

Nanotemplating for two-dimensional molecular imprinting.

A new 2D molecular imprinting technique based on nanotemplating and soft-lithography techniques is reported. This technique allows the creation of target-specific synthetic recognition sites on different substrates using a uniquely oriented and immobilized template and the attachment of a molecularly imprinted polymer on a substrate. The molecularly imprinted polymer was characterized by AFM, fluorescence microscopy, and ATR-FTIR. We evaluated the rebinding ability of the sites with theophylline (the target molecule). The selectivity of the molecularly imprinted polymer was determined for the theophylline-caffeine couple. The molecularly imprinted polymer exhibited selectivity for theophylline, as revealed by competitive rebinding experiments. Fluorescence microscopy experiments provided complementary proof of the selectivity of the molecularly imprinted polymer surfaces toward theophylline. These selective molecularly imprinted polymers have the potential for chemical sensor applications. Because of its 2D nature, this novel chemical sensor technology can be integrated with many existing high-sensitivity multichannel detection technologies.

Neurogenesis and neuronal communication on micropatterned neurochips.

Neural networks are formed by accurate connectivity of neurons and glial cells in the brain. These networks employ a three-dimensional bio-surface that both assigns precise coordinates to cells during development and facilitates their connectivity and functionality throughout life. Using specific topographic and chemical features, we have taken steps towards the development of poly(dimethylsiloxane; PDMS) neurochips that can be used to generate and study synthetic neural networks. These neurochips have micropatterned structures that permit adequate cell positioning and support cell survival. Within days of plating, cells differentiate into neurons displaying excitability and communication, as evidenced by intracellular calcium oscillations and action potentials. The structural and functional capacities of such simple neural networks open up new opportunities to study synaptic communication and plasticity.

A general synthetic route to indenofluorene derivatives as new organic semiconductors.

A series of 2,6-disubstituted indenofluorene derivatives were obtained in high purity via a general route involving the Suzuki coupling reaction. The potential of these conjugated indenofluorenes as new organic semiconductors was demonstrated by the light-emitting diode reaching a high luminance of 1400 Cd/m(2) below 10 V. [structure: see text]