An ultrasensitive sandwich-type electrochemical immunosensor for PSA detection: employing layer-by-layer self-assembly with poly(thionine)/carbon nanotube multilayers as electron mediators and hemin-functionalized PEI/SiO2 nanoparticles as signal amplifiers

Abstract

In this study, hemin-functionalized polyethyleneimine/silica nanoparticles (hemin/PEI/SiO₂ NPs) served as an electrochemical label for ultrasensitive detection of prostate-specific antigen (PSA), a clinically important prostate cancer biomarker. A poly(thionine)/carbon nanotube (PTh/CNTs) multilayer film was fabricated on a glassy carbon electrode (GCE) via integrated electropolymerization and electrostatic layer-by-layer (LbL) assembly, forming a {PTh/CNTs}₂/PTh modified GCE with abundant amino groups. PSA capture antibodies (Ab₁) were then covalently immobilized on the modified electrode through glutaraldehyde (GA) crosslinking, leveraging the film's surface-exposed amino groups. Furthermore, hemin/PEI/SiO₂ NPs were synthesized through amine-carboxyl interactions between carboxyl groups of hemin and amine groups of PEI/SiO₂ NPs. The immunosensor assembly employed a sandwich-type electrochemical platform using PSA detection antibody (Ab₂)-conjugated hemin/PEI/SiO₂ NPs. The specific antibody-antigen binding enables efficient electron transfer across the nanoscale spacing (several nanometers) between hemin/PEI/SiO₂ NPs and the {PTh/CNTs}₂/PTh multilayer film. In this architecture, CNTs serve as excellent electron mediators, significantly facilitating electron transport. Moreover, the high-density immobilization of hemin on PEI/SiO₂ NPs significantly amplifies the electrochemical redox currents of the multilayer film through proximity effects. This enhanced electrical signal enables highly sensitive detection of target protein antigen–antibody binding events. Consequently, by correlating the enhanced reduction peak current of the immunosensor with PSA concentration, highly sensitive detection of PSA at various concentrations can be achieved. Under optimized conditions, it exhibited a linear response to logarithmic PSA concentrations (0.01 pg mL⁻¹ to 100 pg mL⁻¹, R = 0.9963), with an ultralow detection limit of 3.0 fg mL⁻¹ (S/N = 3). Furthermore, the immunosensor demonstrated outstanding analytical performance in human serum samples (96.0–103% recovery, n = 3), confirming its high accuracy for clinical applications.