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Abstract
Porous silicon (PSi) photonic crystals have aroused interest as chemical and biological sensing devices and have been used as label-free optical biosensors in recent years. The advantage of using PSi as a sensor comes from the ease of fabrication and high quality optics. The recent advances in modifying the surface of these porous materials with different surface chemistries have been utilized to add stability to the otherwise unstable freshly etched PSi surface. The results presented in this thesis have been obtained while developing porous silicon microsensors for their use in detecting protease enzyme activity in vivo. The research presented here primarily focuses on the additions made to the surface modification strategies to add functionalities to the surface for specific target (enzyme) detection and quantification both in vitro and in vivo.
A specific class of photonic crystals, rugate filters were fabricated on p-type silicon and used as transducers to transform biological changes to easily read-out optical signals. The presence of a high reflectivity resonant stop-band in the reflectance spectra of these filters made them an ideal choice to be used as a transducing element thereby adding sensitivity to the biosensor. A chemical route based on hydrosilylation of alkynes was adopted to modify the freshly etched PSi surface. This was followed by the copper (I) - catalysed azide cycloaddition reaction using a synthetic anti-fouling polymer to resist non-specific adsorption of biomolecules. Immobilisation of enzyme responsive peptides to these organic layers added specificity to the PSi structure. This specificity was demonstrated by the selective detection of matrix metalloproteases (MMP), MMP-2 and MMP-9 enzymes released from different primary ocular cells by measuring optical blue shifts of these PSi rugate filters. Detection of pg quantities of these MMPs was achieved using these microsensors. The sensitivity achieved by the PSi microsensors was lower than that achieved with zymography; the standard protease detection technique. This functionalisation of the surface with peptides adds versatility to the surface and allows for a range of other biological species to be detected for applications in biology and medicine.
Towards protease detection in vivo, PSi microparticles were injected into eyes of live rabbit animal models. The microparticles were shown to be biocompatible for up to 4 weeks. The animal models were injected with a bacterial endotoxin, lipopolysaccharide to cause endotoxin induced uveitis (EIU) with associated up regulation of the release of proteases inside the eye. Efforts were made to detect the release of MMP-9 in vivo by the injected PSi microparticles by measuring optical changes in the reflectivity spectra of these particles in real time. Digestion of the peptide was detected down to 1.3 picomoles (11.7 nM) of the protease in the uveitis infected eye.
The detection of proteases both in vitro and in vivo via the use of robust, selective and flexible surface chemistry on PSi photonic crystals demonstrates the ability of these photonic structures to be used in biological assays and diagnosis of diseases.