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This paper describes the integration of opto-chemosensors in microfluidics networks. Our technique exploits the internal surface of the network as a platform to build a sensing system by coating the surface with a self-assembled monolayer and subsequently binding a fluorescent sensing molecule to the monolayer. Fluorescent molecules were used that can switch between a fluorescent and a non-fluorescent state, depending on the acidity of the surrounding solution. Two systems were investigated. The first employs surface confinement of a Rhodamine B dye in a glass micro channel that serves as a molecular switch in organic solutions. Upon rinsing the micro channels with acidic or basic solutions it was possible to switch between the fluorescent and non-fluorescent forms reversibly. Moreover, this system could be used to monitor the mixing of two solutions of different acidity along the micro channel. To widen the scope of optical sensing in micro channels an Oregon Green dye derivative was immobilized, which functions as a sensing molecule for pH differences in aqueous solutions. In this case, a hybrid system was used consisting of a glass slide and PDMS channels. The fluorescence intensity was found to be directly correlated to the pH of the solution in contact, indicating the possibility of using such a system as a pH sensor. These systems allow real-time measurements and can be easily implemented in micro- and nanofluidics systems thus enabling analysis of extremely small sample volumes in a fast and reproducible manner.

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The influence of neuron-adhesive pattern geometry on long-term adhesion, survival and pattern compliance of cortical neuronal tissue was studied over a period of 15 days. The results are relevant for a successful, long-term integration of neuronal cells with electrodes from micro-electronic devices. Microwells (depth 0.5 microm), with diameters of 25, 50, 100 and 150 microm and spacing distances of 15, 30, 60 and 90 microm, were etched in a neuron-repellent fluorocarbon (FC) layer and coated with neuron-adhesive polyethylenimine (PEI). Results showed that adhesion, survival and compliance to the underlying patterns were geometry- and time-dependent. After 1 day, adhesion was inversely proportional to the diameter of the microwells, thus favouring the 25 microm microwells. However, adhesion was best on 50 microm microwells after 15 days. Survival of neurons was limited on 25 microm microwells (viability function V(D, T) was 0.08), as opposed to the better survival on 150 microm microwells (V(D, T) was 0.25) after 15 days. In summary, the study shows that the chemical patterns with microwells of 150 microm diameter (90 microm spacing gap) are most suitable for application on neuro-electronic devices owing to the better long-term survival and high pattern compliance of the neuronal cells.

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Adhesion and patterning of cortical neurons was investigated on isolated islands of neuron-adhesive polyethylenimine (PEI) surrounded by a neuron-repellent fluorocarbon (FC) layer. In addition, the development of fasciculated neurites between the PEI-coated areas was studied over a time period of fifteen days. The patterns consisted of PEI-coated wells (diameter 150 microns, depth 0.5 micron) which were etched in a coating of fluorocarbon (FC) on top of polyimide (PI) coated glass. The separation distance between the PEI-coated wells were varied between 10 and 90 microns. This paper shows that chemical patterns of PEI and FC result in highly compliant patterns of adhering cortical neurons after one day in vitro. Interconnecting neurite fascicles between PEI-coated wells were especially present on patterns with a separation distance of 10 microns after eight days in vitro. A significant lower number of interconnecting neurite fascicles was observed on 20 microns separated patterns. Effective isolation of neurons into PEI-coated wells was achieved on patterns with a separation distance of 80 microns as no interconnecting neurite fascicles were observed.

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Entellan is a good sealing substance for culture chamber walls and does not interfere with the growth of the cultured DRG cells. If the Entellan becomes brittle the glass ring can easily be loosened by placing the chamber with the sealed ring for 24 hrs in xylene. Repeated rinsing with xylene and subsequently with hydrated steps of ethanol (100%-70%-30%) allows the ring-wall and chamber floor with its electrodes to be reused.

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