Post‐Imprinting‐Modified Molecularly Imprinted Nanocavities with Two Synergetic, Orthogonal, Glycoprotein‐Binding Sites to Transduce Binding Events into Fluorescence Changes

Abstract We developed sensitive and selective polymeric nanocavities for glycoprotein detection using a novel molecular imprinting technique involving post‐imprinting modification (PIM). It allowed the introduction of two different interaction sites and a fluorescent reporter within each nanocavity. The hepatocellular carcinoma biomarker α‐fetoprotein (AFP) was used as a model glycoprotein. For PIM‐based molecular imprinting, AFP conjugated with polymerizable groups by disulfide bonding was prepared and immobilized on a 4‐carboxy‐3‐fluorophenylboronic acid‐immobilized substrate by cyclic diester formation between AFP glycans in an oriented immobilization manner, facilitating the creation of homogeneous AFP‐imprinted nanocavities. Surface‐initiated controlled/living radical polymerization was performed with a functional monomer, co‐monomer, and crosslinker. The disulfide bonds and cyclic diesters were cleaved to create AFP‐imprinted nanocavities, generating free thiol groups present inside the nanocavities only. A thiol‐reactive fluorescent dye was added to the nanocavities by in‐cavity PIM, yielding signaling AFP‐imprinted nanocavities capable of transducing AFP‐binding events into fluorescence changes. Reference proteins showed little response, while the limit of detection of AFP was 0.27 ng/mL (ca. 3.9 pM) in diluted human serum. Thus, the proposed in‐cavity PIM‐based molecular imprinting technique is effective for ELISA‐relevant, antibody‐free sensing systems for glycoproteins.