Electrochemical biosensor based on Novozym 435 enzymatic ring-opening polymerization for CEA detection

Carcinoembryonic antigen (CEA) is a broad-spectrum tumour marker, with its concentration levels serving as a crucial predictor of cancer diagnosis. In this study, we synthesized a polycaprolactone (PCL) macromolecular polymer via enzyme ring-opening polymerization (eROP). This polymer was then utilized as a signal amplification element in the “sandwich structure” electrochemical impedance biosensor designed for CEA detection. Initially, 3-mercaptopropionic acid (MPA) was self-assembled onto the electrode surface via a gold-sulfur bond. The carboxyl terminus of MPA was then activated using carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS). Subsequently, antibody 1 (Ab1) was immobilised on the electrode surface through the formation of an amide bond, serving as a recognition probe. To prevent non-specific binding, the remaining sites were blocked with Bovine Serum Albumin (BSA). Following the specific capture of CEA by Ab1, the unreacted amino group on the electrode surface was sealed using acrolein. Antibody-2 (Ab2) was then introduced to specifically recognize CEA, forming a classic antibody–antigen–antibody “sandwich structure.” Finally, a DMPA (2,2-dihydroxymethylpropionic acid)-PCL (Polycaprolactone) polymer was conjugated to the electrode surface as a signal amplification unit. The impedance signal strength was subsequently measured using Electrochemical Impedance Spectroscopy (EIS). Under optimal conditions, the biosensor demonstrated a wide linear range (1 pg mL⁻¹–100 ng mL⁻¹) and a low detection limit of 0.38 pg mL⁻¹. The sensor also exhibited high selectivity, stability and reproducibility when tested with clinical serum samples, highlighting its potential applications for early diagnosis and clinical monitoring.