Bio-microdevices are miniaturised devices based on biologically derived components
(e.g., DNA, proteins, and cells) combined or integrated with microfabricated substrates.
These devices are of interest for numerous applications, ranging from drug discovery, to
environmental monitoring, to tissue engineering. Before a bio-microdevice can be fully
developed, specific fabrication issues need to be addressed. One of the most important
is the spatial immobilization of selected biomolecules in specific micro-areas of the
device. Among the biomolecules of interest, the controlled immobilization of proteins to
surfaces is particularly challenging due to the complexity of these macromolecules and
their tendency to lose bioactivity during the immobilization step. The present Thesis
reports on three novel micropatterning techniques for the spatial immobilization of
proteins with bioactivity retention and improved read-out of the resulting micropatterns.
The technologies developed are based on three different micropatterning approaches,
namely 1) direct-writing UV laser microablation (proLAB), 2) a novel microcontact
printing method (�CPTA) and 3) a replica molding method combined with bead selfassembly
(BeadMicroArray). The first two technologies, proLAB and �CPTA, are an
implementation of existing techniques (laser ablation and �CP, respectively), whereas
the third, i.e., the BeadMicroArray, is a totally new technique and type of patterning
platform.
'ProLAB' is a technology that uses a micro-dissection tool equipped with a UV laser
(the LaserScissors�) for ablating a substrate made of a layer of ablatable material, gold,
deposited over a thin polymer layer. The latter layer is transparent to the laser but
favours protein adsorption. In the present work microchannels were chosen as the
structure of interest with the aim of arranging them in 'bar-codes', so to create an
'information-addressable' microarray. This platform was fabricated and its application
to specific antigen binding demonstrated.
The second technique that was developed is a microstamping method which exploits the
instability of a high-aspect ratio rubber stamp fabricated via soft-lithography. The
technique is denominated microcontact printing trapping air (�CPTA) since the collapsing of a rubber stamp made of an array of micro-pillars over a plane glass surface
resulted in the formation of a large air gap around the entire array. The method can be
successfully employed for printing micro-arrays of proteins, maintaining biological
activity. The technique was compared with robotic spotting and found that microarrays
obtained with the �CPTA method were more homogeneous and had a higher signal-tonoise
ratio.
The third technique developed, the BeadMicroArray, introduces a totally new platform
for the spatial addressable immobilization of proteins. It combines replica molding with
microbead self-assembling, resulting in a platform where diagnostic beads are entrapped
at the tip of micropillars arranged in a microarray format. The fabrication of the
BeadMicroArray involves depositing functional microbeads in an array of V-shaped
wells using spin coating. The deposition is totally random, and conditions were
optimised to fill about half the array during spin coating. After replica molding, the
resulting polymer mold contains pyramid-shaped posts with beads entrapped at the very
tip of the post. Thanks to the fabrication mode involved, every BeadMicroArray
fabricated contains a unique geometric code, therefore assigning a specific code to each
microarray. In the present work it was demonstrated that the functionality of the beads
after replica molding remains intact, and that proteins can be selectively immobilized on
the beads, for instance via biorecognition. The platform showed a remarkable level of
selectively which, together with an efficient blocking towards protein non-specific
adsorption, lead to a read-out characterized by a very good signal-to-noise. Also, after
recognition, a code was clearly visible, therefore showing the encoding capacity of this
unique microarray.
| Identifer | oai:union.ndltd.org:ADTP/216592 |
| Date | January 2006 |
| Creators | Filipponi, Luisa, n/a |
| Publisher | Swinburne University of Technology. |
| Source Sets | Australiasian Digital Theses Program |
| Language | English |
| Detected Language | English |
| Rights | http://www.swin.edu.au/), Copyright Luisa Filipponi |
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