Low-fouling SPR detection of lysozyme and its aggregates

Abstract

Protein aggregates adsorb to material surfaces in a different manner than protein monomers and pose additional challenges for biosensor development with regard to non-specific adsorption (NSA). In this context, we describe herein the performance of a new antifouling thiol in a sensor coating resistant to NSA from lysozyme monomers and aggregates. Coatings were prepared as mixed self-assembling monolayers (SAMs) using a long polyethylene glycol carboxyl-terminated thiol (“PEG-COOH”) for the first time in conjunction with a shorter monoethylene glycol hydroxyl-terminated diluent thiol (“MEG-OH”). SAMs and their antifouling properties were characterized by a variety of surface analysis techniques. A key result was that the cleaning procedure drastically affects the antifouling properties of resulting MEG-OH based SAMs. Mixed PEG-COOH/MEG-OH SAMs formed on borohydride cleaned interfaces are able to reduce lysozyme NSA by >90% compared to bare gold; a remarkable performance also displayed for oligomers regardless of their stage of aggregation. Gratifyingly, subsequent SAM functionalisation with an anchoring layer of neutravidin for the preparation of a lysozyme sensor did not significantly alter the antifouling properties of the resulting assembly. The limit of detection for monomeric lysozyme by surface plasmon resonance was 0.3 μg mL⁻¹ with a dynamic range of 3–50 μg mL⁻¹ (R² = 0.9993). The sensitivity of the technique for the aggregated lysozyme was almost two times higher than that for the protein monomer.