Cellular patterning plays an essential role in the development of cell-based biosensors, cell culture analogues and tissue engineering. In particular addressability at cell level is needed for the recording of neuronal activity and interpretation of neuronal communication, thus providing insight in learning and memory processes as well as in the influence of drugs on neuronal activity.
In this paper, we report an approach to guide neuronal growth into simplified networks with single cell resolution. This enables direct correlation of individual neurons with relevant positions on a chip surface and can improve activity recordings by means of microelectrodes1 or field-effect transistors. Our method is based on aligned micro contact printing and allows the deposition of different types of guidance cues with micrometer precision in an easy manner. Making use of a flipchip bonder, we have created patterns of poly-L-lysine (PLL) and laminin to guide the development of neuronal networks. To ensure long-term stability of a neuronal pattern, chemical guidance cues alone do not suffice. Cell compliance to the cytophilic pattern seldomly extends beyond the duration of one week in culture. We have therefore made use of the hydrophobic properties of fluoropolymers such as teflon to discourage neuronal adhesion and at the same time create topography to hold the neurons mechanically in place. This cytophobic pattern was complemented with an aligned deposition of PLL between the teflon lanes. In this paper, aligned micro contact printing, combined chemical and topographical functionalisation of surfaces and the results of the evaluation in primary hippocampal cultures by immunocytochemical staining will be discussed.
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.