A Sequential Epitope-Protein Imprinting Strategy for Antibody-Free Electrochemical Sensing of CD44 Glycoprotein with Ultralow Detection Limit

Developing molecularly imprinted polymer-based sensors for proteins remains challenging due to the complexity, large molecular size, and conformational flexibility of protein targets. Here, we report a novel sandwich-type electrochemical sensor fabricated through a sequential epitope-protein imprinting strategy. This approach employs a two-step ″epitope-first-then-protein″ workflow: peptide epitopes are first site-specifically immobilized onto functionalized magnetic nanoparticles via metal-mediated coordination to form epitope-imprinted probes, which are then complexed with the full protein and serve as templates for protein-level imprinting on the electrode surface. This stepwise imprinting process, transitioning from peptide-level to protein-level recognition, enables the formation of highly tailored molecular cavities that complement the target protein both morphologically and functionally. The sensor synergistically combines the advantages of epitope imprinting (high selectivity, ease of handling) and protein surface imprinting (structural integrity). Integrated with a customized, 3D-printed miniaturized electrochemical cell, the sensor achieves an exceptionally broad linear dynamic concentration range from 0.5 pg·mL⁻¹ to 200 ng·mL⁻¹ and an ultralow detection limit of 9.98 fg·mL⁻¹ (S/N = 3) for the CD44 transmembrane glycoprotein in a 10 μL sample volume. It also exhibits excellent selectivity against common serum proteins, outstanding reproducibility (RSD = 1.6%, n = 7), and robust stability (retaining >90% activity after 20 days). Real-sample analyses in human saliva and mouse serum validate the sensor's high accuracy and applicability in complex biological matrices, demonstrating the potential of this strategy as a generalizable platform for clinical diagnosis of protein biomarkers.