Immobilized Antibodies on Mercaptophenylboronic Acid Monolayers for Dual-Strategy Detection of 20S Proteasome

Abstract

A dual strategy for the electrochemical detection for 20S proteasome (20S) is proposed, based on the oriented immobilization of a capture monoclonal antibody (Abβ) on a self-assembled monolayer of 4-mercaptophenylboronic acid (4-MPBA) on gold electrodes, which led to the Au/4-MPBA/Abβ immunosensor. The methodology comprises the correlation of 20S concentration with (i) its proteolytic activity toward the Z-LLE-AMC substrate, using the Au/4-MPBA/Abβ/20S, and (ii) the enzymatic activity of an alkaline phosphatase (AlkP) from the AlkP-labeled secondary antibody (Abcore-AlkP), which involves the conversion of aminophenylphosphate to the electroactive aminophenol using Au/4-MPBA/Abβ/20S/Abcore-AlkP. The step-by-step construction of the immunosensor and the interactions at its surface were evaluated by surface plasmon resonance and gravimetric analysis with quartz crystal microbalance, showing a high affinity between both antibodies and 20S. Morphological analysis by scanning electron microscopy demonstrated a pattern of parallel lines upon immobilization of Abβ on 4-MPBA and morphological changes to a well-organized granular structure upon binding of 20S. A voltametric and impedimetric characterization was performed after each step in the immunosensor construction. The two detection strategies were evaluated. It was shown that the immunosensor responds linearly with 20S concentration in the range between 5 and 100 µg mL−1, which corresponds to proteasome levels in serum in the case of diverse pathological situations, and LoD values of 1.4 and 0.2 µg mL−1 were calculated for the detection strategies. The immunosensor was applied to the detection of 20S in serum samples with recovery values ranging from 101 to 103%.

Recent Advancements in Receptor Layer Engineering for Applications in SPR-Based Immunodiagnostics

“… ( a ) The architecture of the oligopeptide SAM-based SPR immunosensor, ( b ) the mechanism of antibody immobilization through the interaction of carbohydrate residue with boronic acid SAM. Adapted with permission from Sankiewicz et al [ 70 ] (Copyright © (2018) Elsevier) and Barsan et al [ 71 ] (under the terms and conditions of the Creative Commons CC BY License). …”

“…Such a configuration allows the appropriate spatial orientation of the immunoreceptors on the surface to be maintained [ 73 ]. SAMs composed of 4-mercaptophenylboronic acid (4-MBA) formed directly on gold [ 71 ] and 3-aminophenylboronic acid, immobilized on modified SPR transducers using graphene oxide carboxyl groups, have been described as compounds directly responsible for the capture of antibodies [ 74 ]. Additionally, amine-terminated ligands can be employed for carbohydrate-mediated immobilization through the previous oxidation of polysaccharide residues to aldehydes, followed by Schiff base formation [ 75 ].…”