Patricia Connolly

An Extracellular microelectrode Array for monitoring electrogenic cells in culture

This paper describes a planar array of microelectrodes developed for monitoring the electrical activity of cells in culture. The device allows the incorporation of surface topographical features in an insulating layer above the electrodes. Semiconductor technology is employed for the fabrication of the gold electrodes and for the deposition and patterning of an insulating layer of silicon nitride. The electrodes have been tested using a cardiac cell culture of chick embryo myocytes, and the physical beating of the cultured cells correlated with the simultaneous extracellular voltage measurements obtained. It was found that extracellular stimulation of the cells was possible via the same electrodes used for recording.

Micropatterned substratum adhesiveness: a model for morphogenetic cues controlling cell behavior.

It is generally considered that tracks of cell adhesiveness are important in controlling cell migration during the development and regeneration of many tissues. In order to investigate this experimentally, a number of techniques have in the past been employed to make patterns of differential adhesiveness for in vitro studies. However, practical limitations on patterning resolution and the introduction of residual topography to the experimental substrata have restricted their usefulness. Here we describe a simplified photolithographic technique for patterning cell adhesiveness which allows a high degree of flexibility and precision. We have quantified, using adhesion and spreading characteristics of BHK cells, the differential adhesiveness that can be created on patterned surfaces, how this alters with the duration of exposure to serum proteins, and how this, in turn, relates to the persistence of cell patterning despite increases in cell density. We believe that this technique will prove extremely useful for the detailed in vitro examination of the mechanisms controlling cell behavior as it offers a degree of precision and ease of fabrication that has previously been unavailable.

Clinical diagnostics opportunities for biosensors and bioelectronics.

The clinical diagnostic market represents a unique opportunity for the introduction of biosensors on a widespread commercial basis. To date, however, very few successful biosensors have been launched in this field or any other, despite many promising ideas. The need for biosensors in this field must be analyzed more critically, to assess in which direction the developing biosensor technology can be most effectively aimed and which areas need a more focused approach from basic research in bioelectronics. The diagnostics market is already a highly competitive field; from an industrial perspective, some of the critical issues that need to be taken into consideration when evaluating biosensor projects are: costs per test, regulatory requirements, quality control, instrumentation design, and test parameter selection.

Novel methods for the guidance and monitoring of single cells and simple networks in culture.

SUMMARY The effects of the topography, adhesiveness and chemistry of surfaces in modulating the behaviour of cells in vivo and in vitro have been extensively researched. However, few natural systems are simple enough to allow straightforward conclusions to be drawn, as many different cues are likely to be present at one time. Microelectronic fabrication, normally employed in making integrated circuits, can produce substrates patterned on scales highly relevant to studies of cell behaviour. In this paper, we describe progress in fabricating simple artificial substrata both at the micrometer and sub-micrometer scales. The former can be considered as models for contact guidance along other cells or axonal processes: the latter, models for guidance along aligned collagen matrices. We have systematically studied the reactions of different cell types to simple cues (steps and grooves). Additionally, it may be possible to produce fine-resolution patterns with differential adhesiveness, or with other cell-specific surface-chemical properties, such as the differential deposition of proteins, e.g. cell adhesion molecules. We also describe early results in using topographic and other cues to guide cells onto patterned metal electrodes, forming simple electrically active networks of controlled design, from which long-term recordings can conveniently be made.

Simultaneous multisite recordings and stimulation of single isolated leech neurons using planar extracellular electrode arrays

Planar extracellular electrode arrays provide a non-toxic, non-invasive method of making long-term, multisite recordings with moderately high spatial frequency (recording sites per unit area). This paper reports advances in the use of this approach to record from and stimulate single identified leech neurons in vitro. A modified enzyme treatment allowed identified neurons to be extracted with very long processes. Multisite extracellular recordings from the processes of such isolated neurons revealed both the velocity and direction of action potential propagation. Propagation in two cell types examined was from the broken stump towards the cell body (antidromic). This was true for spontaneous action potentials, action potentials produced by injecting current into the cell body and extracellular stimulation of the extracted process via a planar extracellular electrode. These results extend previous findings which have shown that the tip of the broken stump of extracted neurons has a high density of voltage-activated sodium channels. Moreover they demonstrate the applicability of extracellular electrode arrays for recording the electrical excitability of single cells.

Single cell mobility and adhesion monitoring using extracellular electrodes

Cell attachment and activity on planar electrodes can be observed in real time by continuously monitoring the electrode impedance. The application of a low frequency a.c. field affects neither the electrode properties nor cell growth. The tightness of the cell/electrode junction can be quantified, and has been shown to be dependent on both cell type and electrode surface. Studies have been carried out on both large cell populations, and the single cell/microelectrode interface.

Finite-element analysis applied to extracellular microelectrode design

Abstract Whilst limited success has been achieved with the use of planar metal microelectrodes to record extracellular action potentials generated by cultured neurons, the signal-to-noise ratios obtained generally make it difficult to identify individual signals accurately. We discuss, with the use of numerical computer solutions, how the electrode design can be modified to achieve significant increases in the signal amplitudes. A variety of electrode geometries in proximity to an ideal spherical cell are modelled by computer, and the potentials at the electrodes calculated using the finite-element method (assuming a uniform transmembrane current density of 10 pA μm −2 ). First, by comparison of the solutions for a cell completely surrounded by conducting fluid with that for a cell above an electrode mounted on a flat insulating base, we illustrate the importance of including extracellular boundaries in the model. We then consider an electrode at the base of a narrow groove cut into the insulator, with the cell confined directly above the electrode, and show how the recorded potential increases significantly with groove depth. Finally, a cell confined in a cubic pit is modelled and the electrode potential found to be greater than seven times that for a planar electrode.

High-resolution esophageal manometry: addressing thermal drift of the manoscan system.

Background  The high resolution esophageal manometry system manufactured by Sierra Scientific Instruments is widely used. The technology is liable to ‘thermal drift’, a change in measured pressure due to change in temperature. This study aims to characterize ‘thermal drift’ and minimize its impact.

Microelectronic and Nanoelectronic Interfacing Techniques for Biological Systems

A new field of research and development has emerged in recent years which relies on the availability of ultra-high precision technology and cross-disciplinary collaboration between electronic engineers, biologists, biochemists and chemists. By exploiting the techniques of photolithography and electron beam lithography devices can be created to interface with biological cells or molecules such as enzymes. This type of work is increasingly referred to under the heading of ‘bioelectronics’ and touches on topics such as biosensors and cell-electrode interfacing.

Opportunities at the skin interface for continuous patient monitoring: a reverse iontophoresis model tested on lactate and glucose

Skin has the potential to provide an important noninvasive route for diagnostic monitoring of human subjects for a wide range of applications. Dimensions of surface features in skin suggest that nanodevices and microdevices could be utilized to monitor molecules and ions extracted from the skin. Methods of enhancing extraction from the skin for diagnostics are being developed including reverse iontophoresis, electroporation and sonophoresis. A model system for the simulation of in vivo extraction of molecules and ions by reverse iontophoresis is described here that displays similar behavior to skin both in terms of molecular flux levels and electrical impedance characteristics. The device has potential for use in the development of complete reverse iontophoresis/sensor systems, allowing sensor and extraction systems to be studied and optimized before being tested in the complex in vivo environment. The system has been tested using glucose and lactate and the results are reported and discussed.