• silicon micromachining
• label-free
• cell cultures
• three-dimensional
• optics

Integration of conjugated luminescent polymers into three-dimensional silicon microstructures for cell culturing and diagnostics

Although in nature cells reside in environments with three-dimensional features, most of what is known about cell proliferation-differentiation-death has been derived from cultures on flat surfaces, such as Petri dishes or glass slides. Investigating and monitoring cell cultures into a three-dimensional matrix has been demonstrated to highlight major effects on cell behaviour, from adhesion and differentiation to apoptosis, that are not similarly evident from cell cultures on flat substrates.
The recent progress in cell culture and microfabrication technologies has suggested the development of a novel generation of label-free cell-based sensors for drug discovery, clinical diagnostics, and detection of toxic agents. Such a new-generation of cell-based biosensors exploits living cells in three-dimensional microstructures as bioreceptors and allows cell morpho-functional changes and/or detachment, induced by exposure to environmental perturbations, to be monitored by a suitable transduction (i.e. optical, electrical) method, possibly in a non-invasive and label-free manner. A number of label-free biosensors exploiting different electrical and optical transduction mechanisms has been reported so far.

The main objective of this thesis is the development of a three-dimensional silicon microstructure for 3D cell cultures and diagnostics. The microstructure consists of a microstructured silicon substrate featuring a two-dimensional array of square holes (size of about 40 micron x 40 micron and depth of about 50 micron) with spatial periods of 70 microns. Anchor structures characterized by a two-dimensional periodicity are integrated between adjacent holes. A photoluminescent and/or electroluminescent conducting polymer film is deposited at the bottom of each hole, so that, upon suitable excitation, the light emitted by the polymer can be used for investigating the biological material. In fact, from the spectral analysis of the light transmitted at each hole through the biological material, it might be possible to identify cells featuring special characteristics, such as tumour cells that have a higher volume and refractive index with respect to healthy cells.

The proposed microstructure has been fabricated by silicon electrochemical micromachining (ECM) technology in a single etching step. ECM advanced features, such as dynamic control of the etching anisotropy (from 1 to 0) as the electrochemical etching progresses, allow the silicon dissolution to be switched in real-time from the anisotropic to the isotropic regime and enable advanced silicon microstructuring to be achieved through the use of high-aspect-ratio functional and sacrificial structures, the former being functional to the microsystem operation and the latter being sacrificed for accurate microsystem fabrication.

We investigated several deposition techniques, ranging from spin-coating to drop-casting and dip-coating. The deposition of the luminescent polymer film on the bottom of the culturing wells is not trivial owing to the very high aspect ratio of these wells.
The polymer film coverage has been investigated via Fluorescence Microscopy and the best results have been reached depositing the polymer by dip-coating.
The device performances have been investigated via optical excitation with a laser emitting within the absorption band of the polymer and collecting, then, the photoluminescence (PL) spectra.

The present work has been focused on the microstructure realization and on the polymer film integration in such structure, but long-term developments could regard the microstructure performance improvement. It is to be highlighted that, even if this is a feasibility study, the results are encouraging for further investigations.

By properly designing the anchor structure, in fact, such structure could act as a two-dimensional photonic crystal. If the photonic crystal structure band-gap falls in the wavelength range of polymer photoluminescence/electroluminescence emission, a confinement of the light emitted by the polymer film in the out-of-plane (perpendicular to the substrate surface) direction is expected.
The resulting reduction of optical losses in the in-plane directions (parallel to the substrate surface) and the increased coupling efficiency with cells living in the holes could improve the microstructure performance.