Patterning neurons-on-chip devices using microcontact printing

12/01/2005

Els Parton, Kurt Winters, Dries Braeken, Koen De Keersmaecker, Carmen Bartic, Gustaaf Borghs, IMEC, Leuven, Belgium

Growing neurons on a chip surface holds great promise for brain research. Recently, a printing technique was developed that allows alignment and combination of different chemical patterns on a chip surface, which can be used to control the position of the neuron on top of the transducers. Also, durable and sensitive enzyme-based field-effect transistors (ENFET) for the selective detection of glutamate, a main excitatory neurotransmitter in the brain that plays a key role in disease mechanisms, were realized in the area of the enzymatic layer of the transducer.

In Leuven, Belgium, a unique alliance has been set up between the nanoelectronics research institute IMEC, the Flanders Interuniversity Institute for Biotechnology, the University Hospital of Leuven, and the Katholieke Universiteit Leuven (K.U. Leuven). Experts in nanoelectronics, surface chemistry, and neurobiology are working together to develop neuroelectronic tools that are useful for the study of brain disorders (for more on neurons-on-chip research, see “Neurons-on-chip for brain research” in the unabridged version of this article at www.solid-state.com).

The Belgian researchers believe in the use of field-effect transistors (FET) for the fast, sensitive, and selective detection of neurotransmitters such as glutamate. Apart from chemical detection, FETs can also be used for the electrical stimulation of neurons and/or detection of their electrical signals. On the chip surface, chemical guidance cues are deposited in combination with topographic structures. When embryonic neurons are seeded onto the structured chip surface, the neurons will be guided to the right positions, indicated by the chemical guidance cues. And thanks to the topography, they will maintain this position over time.

Microcontact printing

Because it is based on elastomeric stamps, the commonly used microcontact printing process is the easiest and least expensive method of applying chemical guidance cues onto a chip surface. To create the stamps, molds are made in photoresist. A PDMS mixture is then cast onto the mold. After 30 min of curing at 110°C, the stamp is gently released from the mold and can then be loaded with chemical compounds. After contact with the substrate, the chemical pattern is transferred to the substrate.

Recently, researchers at IMEC optimized microcontact printing so it can be used to deposit and align different patterns of chemical substances with high precision. The alignment capability was achieved by using alignment markers on both the substrate and the stamp. The stamp was then mounted onto a flip-chip bonder that contains a bilateral camera positioned between the stamp and substrate; the camera allows the superposition of the alignment markers.

The aligned microcontact printing technique was used to deposit a (Stenger) pattern made from two different guidance cues. The first pattern comprises the cell body adhesion sites and interrupted dendrite guidance lanes and is used to deposit poly-L-lysine. A second pattern represents uninterrupted wider lanes for axonal development and is used to deposit a mixture of poly-L-lysine and laminin. This combination of different guidance cues for dendrite and axon growth leads to controlled neuronal polarity (Fig. 1).

Figure 1. Fluorescence microscopy images of a) nonguided neuron growth and b) guided neuron growth. For the latter, chemical guidance cues are used and printed with the aligned microcontact technique.Click here to enlarge image

Using this aligned microcontact printing technique, it is possible to realize a highly structured neuronal network and achieve single-cell resolution - i.e., a single cell body adhering to a single site (Fig. 2). Moreover, the alignment capability of this microcontact printing technique would be very useful to print the chemical substances onto a topographic structured chip surface.

Figure 2. Light microscopy image of a large neuronal network, patterned and printed as described in Fig. 1.Click here to enlarge image

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Teflon topography

Chemical guidance cues can be used to initially steer neuronal growth, but topography is needed to keep the neurons in this position. Usually, relatively high structures are applied to “cage” the cell bodies. In the previously mentioned research, topography was used based on cytophobic material, in this case, Teflon. Neurons are able to move over these low structures, but will not attach to the surface because of the cytophobic properties of the material.

Experiments were set up with Teflon topography and regions of poly-L-lysine printed inside the Teflon structures. The exact positioning of poly-L-lysine in between the topographical structures was made possible by the high-resolution aligned microcontact printing technique. Neurons selectively settled only onto the poly-L-lysine regions.

ENFET for glutamate detection

Researchers at IMEC have also developed an ENFET for detection of glutamate to be integrated in a neuroelectronic system. Such devices could provide a fast, highly sensitive, selective, and nondestructive method for measuring neurotransmitters. An ENFET is actually a MOSFET without gate metallization. Thus, a bare oxide is left, which, in the case of an ENFET, is covered with an enzymatic layer. This layer forms the contact between the electronic device and the neurons.

The enzyme used at IMEC to functionalize the ENFET is glutamate oxidase (GLOD). When glutamate comes in contact with this enzyme, hydrogen peroxide and ammonia are formed. This generates a pH shift in the immediate vicinity of the pH-sensitive gate oxide of the ENFET.

A stable and abundant enzyme loading was achieved on the Ta2O5 gate oxide by using (bottom to top): a self-assembled monolayer, poly-L-lysine, gluteraldehyde as a crosslinker, and then the GLOD enzyme itself. Not only does poly-L-lysine enhance enzyme loading, it is also beneficial for neuron growth and survival.

Response curves showed highly sensitive detection of glutamate with detection limits in the tenths of micromoles range as well as good selectivity.

Conclusion

Neuroelectronic systems will enable brain researchers to enter an exciting era that promises to bring new insights into neuronal communication. However, the link between microelectronics and biology will not be easy to make. Research groups with expertise in different disciplines will need to tackle the main challenges: develop sensitive transducers to measure the neuronal signals, work out methods to position the neurons on the chip surface, and exploit biological phenomena in order to create an appropriate environment on the chip surface to ensure survival of the neurons and sufficient coupling with electronics.

Acknowledgment

This work was authored by Els Parton, with the experiments
esearch performed by Kurt Winters, Dries Braeken, Koen De Keersmaecker, Carmen Bartic, and Gustaaf Borghs. Teflon is a registered trademark of DuPont.

Reference

1. D.A. Stenger, et al., “Microlithographic Determination of Axonal/Dendritic Polarity in Cultured Hippocampal Neurons,” J. Neurosci. Meth., Vol. 82, pp. 167-173, 1998.

For more information, contact Els Parton at [email protected].