Aptamer-enabled Surface-Imprinted Self-Assembled Monolayer for Glycoprotein Detection Based on the in Situ Self-Assembly of Lectin and Glucose Oxidase for Signal Amplification

Dual-recognition elements can strengthen target binding event, avoid non-specific adsorption, and improve analytical accuracy. Surface-imprinted self-assembled monolayer (SAM) formed on the substrate surface by the co-assembly of template molecules and organic monomers is considered as a prospective alternative to the conventional molecularly imprinted polymers. Herein, we propose a strategy for protein recognition through the formation of aptamer-enabled antifouling peptide-imprinted SAM. The aptamer-protein conjugates were anchored on the gold surface, and then antifouling zwitterionic peptides were assembled around the aptamer-protein conjugates to form imprinted SAM. Removing the bound proteins by an acidic solution allowed for the formation of biocompatible cavities for target rebinding. The antifouling peptides could eliminate the non-specific adsorption and strengthen the target binding event through the formation of imprinted cavities. The dual-recognition system was used to directly detect carcinoembryonic antigen (CEA) at the concentration down to 0.1 ng/mL by electrochemical impedance spectroscopy. Furthermore, homodimeric glucose oxidase (GOx) was in-situ assembled on the electrode surface to form protein networks by using homotetramer concanavalin A (ConA) as both the recognition element and the crosslinker, thereby achieving enzymatic signal amplification. The sensitivity was improved by 100-fold through the signal amplification of ConA-GOx assemblies. The proposed strategy opens up a universal route for the design of novel biosensors for the dual-recognition and accurate detection of biomarkers, providing valuable insights into the fabrication of imprinting materials and the development of innovative biosensing platforms.