An electrocatalytic dual molecularly imprinted polymer-based biosensor enables femtomolar detection of CD44 without...

The development of sandwich-type electrochemical immunosensors is critically constrained by the limited availability and high cost of well-matched antibody pairs. To address this, we present an antibody-free biosensing platform for the ultrasensitive detection of the cancer biomarker CD44, utilizing a pair of molecularly imprinted polymers (MIPs). A detection probe (P-MIP) featuring a sub-4-nm imprinted layer is synthesized on magnetic nanoparticles via metal affinity-oriented surface imprinting, leveraging Cu²⁺-histidine coordination for directional template immobilization. A complementary capture probe (E-MIP) is then formed on a miniaturized, 3D-printed electrode by in situ electropolymerization of a nanoscale polydopamine layer around the P-MIP-pre-captured CD44. This dual-orientation imprinting strategy effectively minimizes non-specific binding and enhanced target specificity. Crucially, the embedded Cu²⁺ centers serve both as structural anchors and intrinsic redox reporters, enabling label-free detection and enhancing conductivity. Integrating a 3D-printed microcell, the sensor achieves an exceptional detection limit of 0.31 pg mL^-1 (13.4 fM) and a wide linear range (0.5 pg mL^-1-50 ng mL^-1) using merely 10 μL of sample. It demonstrates remarkable selectivity (<3 % cross-reactivity), high reproducibility (RSD = 1.9 %), excellent stability (90.5 % signal retention over 13 days), and satisfactory recovery rates (96.8-100.1 %) in spiked human saliva and mouse serum. This work establishes a versatile and robust analytical platform for the precise detection of protein biomarkers, holding significant promise for practical diagnostic applications.