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 sensing platforms can strengthen target-binding events, avoid non-specific adsorption, and improve analytical reliability for complex samples. Aptamer has become highly promising recognition element for the development of advanced biosensing platforms. 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 conventional molecularly imprinted polymers. Herein, we propose a strategy for recognition and detection of glycoproteins through the formation of aptamer-enabled antifouling peptide-imprinted SAM. The aptamer-protein conjugates were first anchored on the gold surface, and then antifouling zwitterionic peptides were assembled around the aptamer-protein conjugates. 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 glycoprotein carcinoembryonic antigen (CEA) at a concentration down to 0.1 ng/mL by electrochemical impedance spectroscopy. Furthermore, homodimeric glucose oxidase (GOx) was in-situ assembled on the CEA-bound 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 dual-recognition and accurate detection of biomarkers, providing valuable insights into the fabrication of imprinting materials and the development of innovative biosensing platforms.