Development of a label-free aptasensor for monitoring the self-association of lysozyme

Abstract

A novel aptamer and surface plasmon resonance (SPR)-based sensor was developed for the label-free detection of lysozyme. The aptasensor is characterised by a detection limit of 1 μg mL⁻¹ and a linear range of 5–50 μg mL⁻¹. As an application, we examined the usefulness of the aptasensor for monitoring the early stages of the aggregation of lysozyme. It was surprisingly found that, despite a significant decrease in monomer content during aggregation, the response of the aptasensor for protein solutions aged for 12 hours was similar to that for the fresh protein. To correlate the results obtained with the aptasensor with the composition of lysozyme solutions at various time points, we examined them in detail by atomic force microscopy (AFM), thioflavin T fluorescence, size-exclusion chromatography (SEC) and Matrix Assisted Laser Desorption Ionisation Time of Flight Mass Spectrometry (MALDI-TOF-MS). All methods together indicated that during the initial hours of aggregation, the protein solutions contained small lysozyme oligomers (mainly dimers) and decreasing amounts of monomers. Our results thus suggest that the aptamer also recognizes lysozyme dimers/oligomers. A higher non-specific binding was observed for the aggregated lysozyme at the surface of the aptasensor as compared to the native protein. This was attributed to the hydrophobic patches which are exposed by the unfolded lysozyme and/or oligomer species, allowing for different adsorption and organisation at the surface of the aptasensor. This hypothesis is supported by square wave voltammetry (SWV) studies using solutions of aggregated lysozyme. A higher electrochemical signal due to the direct oxidation of tyrosine/tryptophan residues was observed for aged protein solutions as compared to the fresh solution, indicative of an increased number of such exposed electroactive residues and of overall increased surface hydrophobicity of the protein. Our work presents a label-free lysozyme aptasensor that is useful not only for the detection of the protein monomer but also for observing the onset of aggregation. The approach can be extended to other proteins which are prone to aggregation.