Simultaneous Detection of Two Tumor Marker Proteins Using Dual-Colored Signaling Molecularly Imprinted Polymers Prepared via Multi-Step Post-Imprinting Modifications

Abstract

Abstract We report simultaneous detection of tumor marker proteins using a molecularly imprinted polymer-based fluorescence sensing system, in which prostate-specific antigen (PSA) recognition cavity, labeled with Alexa Fluor 594, and α-fetoprotein (AFP) recognition cavity, labeled with Alexa Fluor 647, exist together in the polymer matrix. The individually fluorescent-labeled PSA- and AFP-imprinted polymer was prepared by a dual imprinting method, followed by multi-step post-imprinting modifications (PIM). A polymerizable group, conjugated with PSA or AFP via a disulfide bond, was prepared and immobilized on a phenylboronic acid moiety-introduced substrate by the formation of cyclic diester between phenylboronic acid and glycans on proteins. The polymer matrix was prepared using surface-initiated atom transfer radical polymerization. After the reduction of the disulfide bond and hydrolysis of the cyclic diester, PSA- and AFP-imprinted nano-cavities were generated simultaneously. In multi-step PIM, thiol-reactive fluorescent dyes were introduced via a dynamic protection procedure using the target protein, which yielded dual fluorescence-labeled imprinted nano-cavities. Fluorescence signaling abilities were assessed, and each AFP and PSA-imprinted nano-cavity was confirmed to transduce the protein binding events into specific fluorescence signals, with lower values of limit of detection (<2.0 ng/mL). Therefore, the proposed methodology could be a novel platform for the simultaneous detection of multiple proteins.