Roeland Huys

Single-cell recording and stimulation with a 16k micro-nail electrode array integrated on a 0.18 μm CMOS chip

To cope with the growing needs in research towards the understanding of cellular function and network dynamics, advanced micro-electrode arrays (MEAs) based on integrated complementary metal oxide semiconductor (CMOS) circuits have been increasingly reported. Although such arrays contain a large number of sensors for recording and/or stimulation, the size of the electrodes on these chips are often larger than a typical mammalian cell. Therefore, true single-cell recording and stimulation remains challenging. Single-cell resolution can be obtained by decreasing the size of the electrodes, which inherently increases the characteristic impedance and noise. Here, we present an array of 16,384 active sensors monolithically integrated on chip, realized in 0.18 μm CMOS technology for recording and stimulation of individual cells. Successful recording of electrical activity of cardiac cells with the chip, validated with intracellular whole-cell patch clamp recordings are presented, illustrating single-cell readout capability. Further, by applying a single-electrode stimulation protocol, we could pace individual cardiac cells, demonstrating single-cell addressability. This novel electrode array could help pave the way towards solving complex interactions of mammalian cellular networks.

Local electrical stimulation of single adherent cells using three-dimensional electrode arrays with small interelectrode distances

In this paper, we describe the localized and selective electrical stimulation of single cells using a three-dimensional electrode array. The chip consisted of 84 nail-like electrodes with a stimulation surface of 0.8 um and interelectrode distances as small as 3 um. N2A cells were used to compare bipolar stimulation between one electrode in- and one outside the cell on the one hand, and two electrodes in the same cell on the other hand. Selective and localized stimulation of primary embryonic cardiomyocytes showed the possibility to use this chip with excitable cells. The response of the cells to applied electrical fields was monitored using calcium imaging whereas assessment of electroporation was determined following influx of propidium iodide. Arrays of these three-dimensional electrodes could eventually be used as a tool to selectively electroporate the membrane of single cells for genetic manipulation or to obtain electrical access to the inner compartment of the cell.

Local electrical stimulation of cultured embryonic cardiomyocytes with sub-micrometer nail structures

In this paper, we demonstrate the feasibility of selective extracellular electrical stimulation at the (sub)cellular level in dissociated cultured cells. Using a CMOS-compatible process, we have fabricated an electrode array with sub-micrometer nail probes. Due to their particular configuration, the nails are strongly engulfed by the cellular membrane. By measuring the calcium signals, we found that electrical stimulation via the micronails activates the cell locally, in a dose-dependent manner, with very low applied currents. The results suggest the applicability of the device in pharmacological or signal propagation studies.

Functionalised microneedles for enhanced neuronal adhesion

Efficient functional coupling of neuronal cells and electronic sensors could result in hybrid bidirectional communication between neurons and computers (L.J. Breckenridge et al., Advantages of using microfabricated extracellular electrodes for in vitro neuronal recordings, J. Neurosci Res. 42 (1995), pp. 266–276; G. Zeck and P. Fromherz, Noninvasive neuroelectronic interfacing with synaptically connected snail neurons immobilised on a semiconductor chip, PNAS 98 (2001), pp. 10457–10462). Such systems could enable us to gain insight into the mechanisms of neuro-degenerative diseases like Parkinsons and Alzheimers disease in vitro or could be used to improve the function and efficiency of devices used in vivo, for example in Deep Brain Stimulation devices that are already used in the treatment of Parkinsons disease. One of the major challenges for the development of reliable neuro-electronic systems is to perform extracellular recordings of action potentials with a high signal-to-noise ratio. The poor quality of these recordings is caused by the culture medium, which is present in the cleft between the cell membrane and the sensor surface (P. Fromherz, Neuroelectronic interfacing: semiconductor chips with ion channels, nerve cells and brain, in Nanoelectronics and Information Technology, R. Waser, ed., Wiley–VCH, Berlin, 2003, pp. 781–810; G. Zeck and P. Fromherz, Noninvasive neuroelectronic interfacing with synaptically connected snail neurons immobilised on a semiconductor chip, PNAS 98 (2001), pp. 10457–10462). In this article, we describe a method allowing a reduction of the distance between the membrane and the surface by combining surface chemistry and topography. We have developed a specialised surface chemistry, based on small laminin-derived peptides, which applied onto the topographical structures, triggers their engulfment by the cell membrane in a phagocytosis-like event. In the phagocytotic pit, the distance between the cell membrane and the sensor surface is believed to be minimal. We describe the surface chemistry used for the controlled immobilisation of the small peptides on the surface of the needle-like structures that are manufactured on the surface of electronic devices. PC12 neuro-blastoma cells and genetically-modified HeLa cells have been used to investigate the interaction between the cell membrane and the peptide functionalised topographical structures. The membrane–surface interaction was examined by means of electron microscopy and fluorescence microscopy.

Micro-sized syringes for single-cell fluidic access integrated on a micro-electrode array CMOS chip

Very-large scale integration and micro-machining have enabled the development of novel platforms for advanced and automated examination of cells and tissues in vitro. In this paper, we present a CMOS chip designed in a commercial 0.18 μm technology with integrated micro-syringes combined with micro-nail shaped electrodes and readout electronics. The micro-syringes could be individually addressed by a through-wafer micro-fluidic channel with an inner diameter of 1 μm. We demonstrated the functionality of the micro-fluidic access by diffusion of fluorescent species through the channels. Further, hippocampal neurons were cultured on top of an array of micro-syringes, and focused ion beam-scanning electron microscopy cross-sections revealed protrusion of the cells inside the channels, creating a strong interface between the membrane and the chip surface. This principle demonstrates a first step towards a novel type of automated in vitro platforms, allowing local delivery of substances to cells or advanced planar patch clamping.