There is real interest in developing selective and sensitive tools to detect protease activity; these play pivotal roles in cancer progression with changes in their amounts and types linked to several pathological processes such as tumour formation, evolution and even suppression. Peptide-based electrochemical assays have been shown to offer several potential advantages over other tools and techniques for development into sensing systems. However, their implantation and use in vivo is complex as they face serious limitations when considering two vital requirements for implantation: sensor miniaturisation for ready implantation and localised measurement and controlled anti-biofouling protection. This study presents the investigation and analysis of these miniaturisation- and protection-related issues and the development of solutions as key steps towards the localised in vivo application and measurements.
The first part of the presented work focuses on the potential for assay miniaturisation. This used commercial platinum microelectrodes which were modified with self-assembled monolayer (SAM)-based protease sensing probes. Building on previous macroelectrode studies, which have explored and optimised the use of different SAM structures, redox labelling, anchor type and various spacers of different lengths, further optimisation was carried out with the aim of developing and defining an optimum microelectrode protocol. Comparison of the quantitative analytical performance of macro- and microelectrode systems established the feasibility of developing miniaturised platforms for efficient and clinically-relevant protease detection. Interestingly, significant differences were observed such as an enhanced reproducibility and decreased cleavage rate for the microelectrodes, which were thought to be indicative of variation in the SAM probe film structure on these electrode surfaces caused by differences in film deposition kinetics. This decreased cleavage (response) rate was mitigated by measurement at normal body temperature which was shown to increase kinetics and suggested the possibility of more rapid in vivo sensing.
These miniaturisation findings on commercial microelectrodes were translated to in-house microelectrodes fabricated as platinum thin film-on-silicon chips. Initial results showed reduced SAM probe stability. As the use of stronger SAM probe anchoring (through tripodanchored probes) did not solve this problem, the underlying reason was attributed to structural differences between the surfaces of commercial and in-house electrodes, resulting in enhanced Pt detachment in the latter. Increasing metal film thickness and post-fabrication annealing did not completely overcome this problem, and the remaining decrease in stability was attributed to increased Pt surface roughness and destabilisation through successive electrochemical oxidation and reduction during acidic cleaning. An alternative electrochemical reductive cleaning method was thus developed and tested on enhanced electrode sensing systems; arrays of microelectrodes (MEA) and microcavity nanoband edge electrodes (MNEE) were fabricated, cleaned using this reductive method, characterised using typical redox couples and then tested for protease sensing. Gratifyingly, these systems were found to be sufficiently reproducible and stable for sensing. Although functionalised MNEEs achieved significantly higher current densities, there was no great enhancement of response rate from decreasing electrode size from micro to nano, consistent with the fact that diffusional transport is not the rate determining step in this cleavage reaction. Given the variability of probe film deposition characteristics and the resulting cleavage rates, the applicability of potential-controlled SAM probe deposition for controlling probe film formation was investigated as a proof-of-concept study.
The second part of this work concentrated on the development of a sensor protection and activation strategy against biofouling. A pH-triggered dissolvable polymeric coating was dropcast onto clean and probe-modified electrodes and then characterised in terms of the delayed activation time, enhancement in anti-biofouling properties and retention of sensing characteristics. These results demonstrated that reproducible delayed sensor activation was achieved and controlled by optimising parameters such as coating thickness, homogeneity and density through the coating method and temperature. Comparative evaluation of polymercoated and uncoated probe-modified electrodes in a biologically relevant medium also revealed significant improvement in their anti-biofouling characteristics
