Carmen Bartic

Neurotoxicity of Alzheimer's disease Aβ peptides is induced by small changes in the Aβ42 to Aβ40 ratio

The amyloid peptides Aβ40 and Aβ42 of Alzheimers disease are thought to contribute differentially to the disease process. Although Aβ42 seems more pathogenic than Aβ40, the reason for this is not well understood. We show here that small alterations in the Aβ42:Aβ40 ratio dramatically affect the biophysical and biological properties of the Aβ mixtures reflected in their aggregation kinetics, the morphology of the resulting amyloid fibrils and synaptic function tested in vitro and in vivo. A minor increase in the Aβ42:Aβ40 ratio stabilizes toxic oligomeric species with intermediate conformations. The initial toxic impact of these Aβ species is synaptic in nature, but this can spread into the cells leading to neuronal cell death. The fact that the relative ratio of Aβ peptides is more crucial than the absolute amounts of peptides for the induction of neurotoxic conformations has important implications for anti‐amyloid therapy. Our work also suggests the dynamic nature of the equilibrium between toxic and non‐toxic intermediates.

Localized electrical stimulation of in vitro neurons using an array of sub-cellular sized electrodes

The investigation of single-neuron parameters is of great interest because many aspects in the behavior and communication of neuronal networks still remain unidentified. However, the present available techniques for single-cell measurements are slow and do not allow for a high-throughput approach. We present here a CMOS compatible microelectrode array with 84 electrodes (with diameters ranging from 1.2 to 4.2 μm) that are smaller than the size of cell, thereby supporting single-cell addressability. We show controllable electroporation of a single cell by an underlying electrode while monitoring changes in the intracellular membrane potential. Further, by applying a localized electrical field between two electrodes close to a neuron while recording changes in the intracellular calcium concentration, we demonstrate activation of a single cell (∼270%, DF/F(0)), followed by a network response of the neighboring cells. The technology can be easily scaled up to larger electrode arrays (theoretically up to 137,000 electrodes/mm(2)) with active CMOS electronics integration able to perform high-throughput measurements on single cells.

Planar 2D-Array Neural Probe for Deep Brain Stimulation and Recording (DBSR)

Implantable micromachined probes with planar electrode arrays for neural stimulation and recording were designed, fabricated and evaluated. Probes have been realized with distributed electrode sites and different electrode configurations (i.e. size and geometry). These were tested for their ability to selectively record and stimulate cortical brain areas. The probes were stereotactically implanted into the cortical region of the rat brains without breaking and were successfully used to measure neural signals and evoke limb contraction in response to electrical stimulation.

In vitro and In vivo electrochemical characterization of a microfabricated neural Probe

The electrochemical behavior of neural implants with 50 µm-diameter platinum electrodes was tested during acute implantations in the motor cortex of anesthetized rats. Custom Ag|AgCl reference electrodes were prepared that could be co-implanted with the probes. The results obtained in vivo are compared with in vitro measurements performed in buffered saline solution (PBS) with and without the addition of bovine serum albumin (BSA). The presence of BSA clearly altered the performance of the electrodes which was studied by means of cyclic voltammetry (CV), electrochemical impedance spectroscopy (EIS), voltage transient measurements (VT) and monitoring of the open circuit potential (OCP). We found that hydrogen gas evolved at 1.22 A/cm2 in BSA-free PBS whereas in BSA-containing PBS it occurred already at 0.51 A/cm2.

Chemical and Biological Characterization of Thiol SAMs for Neuronal Cell Attachment

Cellular adhesion and growth on solid-state surfaces is the central theme in the development of cell-based biosensors and implantable medical devices. Suitable interface techniques must be applied to construct stable and well-organized thin films of biologically active molecules that would control the development of neuronal cells on chips. Peptides such as RGD fragments, poly-L-lysine (PLL), or basal lamina proteins, such as laminin or fibronectin, are often used in order to promote cellular adhesion on surfaces. In this paper we describe the characterization of several self-assembled monolayers (SAMs) for their ability to anchor a laminin-derived synthetic peptide, PA22-2, a peptide known to promote neuronal attachment and stimulate neurite outgrowth. We have evaluated the immobilization of PA22-2 onto 16-mercaptohexadecanoic acid, 4-maleimide-N-(11-undecyldithio)butanamide, and 2-(maleimide)ethyl-N-(11-hexaethylene oxide-undecyldithio)acetamide SAM functionalized Au substrates. The neuronal attachment and outgrowth have been evaluated in embryonic mouse hippocampal neuron cultures up to 14 days in vitro. Our results show that differences in the cell morphologies were observed on the surfaces modified with various SAMs, despite the minor differences in chemical composition identified using standard characterization tools. These different cell morphologies can most probably be explained when investigating the effect of a given SAM layer on the adsorption of proteins present in the culture medium. More likely, it is the ratio between the specific PA22-2 adsorption and nonspecific medium protein adsorption that controls the cellular morphology. Large amounts of adsorbed medium proteins could screen the PA22-2 sites required for cellular attachment.

A novel 16k micro-nail CMOS-chip for in-vitro single-cell recording, stimulation and impedance measurements

In neurophysiological and pharmaceutical research, parallel and individual access to a dense population of in-vitro cultured neurons is a key feature for analyzing networks of neurons. This paper presents a 0.18µm CMOS chip containing a dense array of micro-nail electrodes, a 128×128 sensor/actuator matrix with in-situ differential amplification circuits, pico-Ampere current stimulation, and impedance measurement circuits. Measurements on packaged chips show successful impedance measurements matching the simulation model and electrical recordings of in-vitro cultured cardiomyocytes, correlated with recorded changes in intra-cellular calcium concentrations. This system is a first step towards a high-throughput neuron/chip interface.

Improving neuronal adhesion on chip using a phagocytosis-like event

Efficient integration of neuronal cells and electronic devices could result in hybrid bi-directional communication systems that would enable us to interact at fundamental levels with biological structures and gain insight in the mechanisms governing their functions. Such systems require a very tight coupling between the neuronal cell membrane and the surface of an electronic chip. In this paper we report an approach where the combination of specialized surface chemistry and the manipulation of biological processes, like a phagocytosis-like process, might improve this coupling. As a model, we used coated micro- and nano-sized beads and induced phagocytosis-like events by adding them to cultured cells. The development of the surface chemistry and the results obtained with beads functionalized with a laminin derived peptide are presented.

An SU-8 liquid cell for surface acoustic wave biosensors

One significant challenge facing biosensor development is packaging. For surface acoustic wave based biosensors, packaging influences the general sensing performance. The acoustic wave is generated and received thanks to interdigital transducers and the separation between the transducers defines the sensing area. Liquids used in biosensing experiments lead to an attenuation of the acoustic signal while in contact with the transducers. We have developed a liquid cell based on photodefinable epoxy SU-8 that prevents the presence of liquid on the transducers, has a small disturbance effect on the propagation of the acoustic wave, does not interfere with the biochemical sensing event, and leads to an integrated sensor system with reproducible properties. The liquid cell is achieved in two steps. In a first step, the SU-8 is precisely patterned around the transducers to define 120 μm thick walls. In a second step and after the dicing of the sensors, a glass capping is placed manually and glued on top of the SU-8 walls. This design approach is an improvement compared to the more classical solution consisting of a pre-molded cell that must be pressed against the device in order to avoid leaks, with negative consequences on the reproducibility of the experimental results. We demonstrate the effectiveness of our approach by protein adsorption monitoring. The packaging materials do not interfere with the biomolecules and have a high chemical resistance. For future developments, wafer level bonding of the quartz capping onto the SU-8 walls is envisioned.

L-glutamate detection using a poly-L-lysine coated ENFET

Synaptic transmission in neuronal networks occur on a very short time scale and is highly specific. Fast, sensitive and in situ detection of single neuron L-glutamate release is essential for the investigation of these events under physiological or pathophysiological conditions. Up till now, amperometry with enzyme-modified electrodes has extensively been used to monitor extracellular glutamate release. However, due to in situ signal amplification, ENzyme-modified Field-Effect Transistors (ENFETs) have the advantage of preserving sensitivity and a fast response time when scaled down to micrometer dimensions. We have realized a L-GLutamate OxiDase (GLOD) functionalized FET to be used for glutamate detection in neuronal cultures. Effective and reproducible immobilization of GLOD on the FET active area is achieved by using Poly-L-Lysine (PLL) as a loading matrix. PLL plays a dual role in the assay: on the one hand this molecule serves as a platform for obtaining high enzyme loading and on the other hand it benefits the survival of the neuronal network on the active area of the FET. Both PLL and enzyme immobilization were characterised by quartz crystal microbalance measurements. A much higher enzyme loading has been achieved by this approach compared to immobilization methods without PLL. The enzyme coating has proven to be extremely durable as it keeps its activity for at least 3 weeks as monitored by a colorimetric assay. FET characterisation curves and glutamate response curves of the ENFET are presented.

Single-cell stimulation and electroporation using a novel 0.18 µ CMOS chip with subcellular-sized electrodes

In drug screening and pharmaceutical research, high-throughput systems that are able to perform single-cell measurements are highly desired. Micro-electrode arrays try to answer this need but still suffer from significant drawbacks such as a small amount of electrodes and the inability to address single cells. Here, we present a novel multi-transistor array chip with 16,384 subcellular-sized electrodes based on 0.18 µm CMOS technology. We show that single-cell stimulation is possible by applying voltage pulses on the electrode to stimulate the cells lying on top. Electroporation of the cell membrane is observed using the whole-cell patch clamp technique and fluorescent dye-based live imaging. This technology could be used for high-throughput, single-cell manipulations for the purpose of large-scale drug screening and the investigation of fundamental cell processes.