Properties and tunable nature of electrochemically-grown peptide-based hydrogels at single microelectrodes

Abstract

Microelectrodes possess enhanced mass transport properties which make them desirable over macroelectrodes in point-of-care electrochemical biosensing platforms. Due to their scale, microelectrode biosensors also face a heightened receptiveness to biofouling, often characterised by the nonspecific binding of large molecules and cells to the sensing layer or electrode surface. This ultimately prevents the biosensor from providing an accurate measure of a chemical target. Anti-fouling, semi-permeable membranes are employed to prevent biofouling at point-of-care biosensors. Recently, peptide-based supramolecular hydrogels have been considered as anti-fouling membranes which can encapsulate and immobilise sensing layers. A pH shift generated by the electrochemical oxidation of hydroquinone triggers the self-assembly of dissolved gelator molecules local to the electrode surface, and the peptide-based gelator molecules aggregate into fibre-like structures that entangle to form the gel in these localised acidic regions.

This thesis first explores the extent to which Carb-Ala hydrogels can be tuned at single 25 μm diameter Pt disc microelectrodes under potentiostatic control. Altering the time and potential applied has allowed for the production of gels with different sizes and densities using the same stock gelator solution, identified by pairing optical microscopy (both in situ and ex situ) and electrochemical impedance spectroscopy techniques. In doing so, a dynamic process between gel assembly and disassembly has been demonstrated: the gels first undergo a loss in density before a collapse in structure, characterised by a gradual increase in transparency and a gradual increase in the diffusion coefficients of a ferro/ferricyanide redox probe.

This process was first observed during continuous gel growth, likely caused by a shift in the dominance of gelator deprotonation and network disassembly (driven by bulk pH 8 gelator solution flux) over self-assembly (driven by proton generation via hydroquinone oxidation). The gels underwent a similar loss in density followed by structure collapse in buffered pH 4.3 and unbuffered pH 0 solutions, though the rate of gel loss was decreased in the lower pH conditions. The phenomenon was also shown not be exclusive to Carb-Ala, as electrochemical gels of a second peptide-gelator BrAV also underwent these changes on a similar timescale. These observations suggested that gel instability might be driven by a concentration gradient of weakly-bound gelator at the electrode surface and the bulk gelator-less solutions.

Lastly, a simple use-case for these gels as enzyme immobilising layers in a 1st generation enzymatic biosensor is presented. To avoid enzyme leaching with the breakdown of gels, electropolymerisation of Carb-Ala post-gelation was utilised as a way of stabilising the preformed, tuned microscale gels. The resulting GOx/Carb-Ala polymer-functionalised glucose biosensors were capable of producing progressively greater signals in the presence of increasing concentrations of glucose (due to the electrochemical oxidation of the H2O2 produced by the enzymes), and the calibration curves of each electrode displayed characteristic Michaelis-Menten kinetics. The sensors possessed a biologically relevant linear calibration range of 0-4 mM, a maximum sensitivity of 189 ± 9 μM mM-1 cm2, a LOD of 0.33 mM, and a t1/2 of up to 44 days at 37 °C.