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

TL;DR

This paper describes a new electrochemical sensor that can detect blood clotting factors to monitor anticoagulant therapy.

Contribution

A novel graphene-based immunosensor is developed to distinguish active and inactive forms of Factor X for DOAC monitoring.

Findings

The sensor successfully differentiated FXa from FX using electrochemical methods with high sensitivity.

Structural analysis confirmed FXa lacks an activation peptide, affecting its electrochemical response.

In silico docking showed the sensor is effective across genetic variations in Factor X.

Abstract

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
pc: −241.4 μA, 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.

Figures