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.

Label-free detection of lysozyme in wines using an aptamer based biosensor and SPR detection

“…Despite these differences, the net result is the same, a high disturbance of lysozyme interaction with its specific aptamer. The effect of salt as observed by SPRreduced binding of lysozyme by the aptamer with increase in ionic strength -was previously documented by us and others [8,32].…”

“…Aptamer immobilization through neutravidin-biotin affinity interaction was reported to be a good approach leading to high sensor specificity and sensitivity [23] and was also successfully used in previous studies conducted in our lab [8,19]. Therefore, we have chosen to functionalize the sensors with neutravidin as per a previously described procedure [8].…”

“…Therefore, we have chosen to functionalize the sensors with neutravidin as per a previously described procedure [8]. We varied the amount of immobilized neutravidin by varying the time allowed for flowing a 10 g/mL protein solution on the sensor at 50 L/min.…”

“…After rinsing several times with water and ethanol and drying under a gentle stream of nitrogen, the interfaces were immersed in a 50 M "PEG-COOH" ethanolic solution for three days. Next, the interfaces were mounted on the SPREETA apparatus and functionalized with Neutravidin by carbodiimide chemistry as described in [8]. A biotinylated aptamer was then immobilized by high affinity capture on one channel (further on designated as "specific" channel), while the second channel was left unmodified, being used to monitor the non-specific binding events (Fig.…”

“…the standard method provided by OIV in 2010) [6] or on immunosensing by ELISA [7]. Various sensors have been described also as alternatives to these classic analytical methods, based mainly on electrochemical and optical methods [8][9][10][11][12][13][14]. Some allowed reaching very low detection limits, down to 10 −14 M [14].…”