Development of an Electrochemical Immunosensor for Detecting Coagulation Factor Xa and Perspectives in Monitoring Direct Oral Anticoagulant Therapy

This study aims to develop a point-of-care (POC) electrochemical immunosensor to monitor the anticoagulant activity of direct oral anticoagulants (DOACs) by detecting and distinguishing between the inactive (FX) and active (FXa) forms of Factor X. DOACs have transformed the management of thromboembolic disorders by selectively inhibiting FXa. Common monitoring methods are either insufficiently sensitive to DOAC levels or not widely accessible. This study demonstrates the development of a graphene-based electrochemical immunosensor modified by gold nanostructures and functionalized with anti-FX antibodies. Electrode modifications were characterized by cyclic voltammetry (CV), differential pulse voltammetry (DPV), and field-emission scanning electron microscopy (FESEM), showing enhanced electron transfer and increased current peaks (<i>I</i> _pc: -241.4 μA, <i>I</i> _pa: 244.6 μA). The sensor distinguished synthetic blood samples with FXa from negative and positive for FX (only 20 μL sample) via DPV peak area analysis (cutoff of 10.65 and 12.42 μA·V). An enzyme-linked immunosorbent assay was performed to verify antigen-antibody interactions, demonstrating less sensitivity to differentiate FX from FXa than DPV. Structural bioinformatics confirmed the loss of a 51-amino acid activation peptide in FXa, reducing molecular volume and likely affecting electrochemical response. In silico docking of seven nonpathogenic F10 variants in DOAC binding regions showed minimal impact on drug binding, supporting sensor applicability across genetic variations. This work demonstrates a sensitive, selective immunosensor for discriminating coagulation factor activation states, supporting point-of-care anticoagulant therapy monitoring.