Detection of factor Xa activity using giant magnetoresistive biosensors

Background: As anticoagulants are widely used to treat patients with atrial fibrillation (AF) and other thrombotic conditions, it is necessary for physicians to determine whether the medication has been taken in emergencies. Among many anticoagulants, rivaroxaban has attracted attention due to its safety and convenience. Since rivaroxaban inhibits activated coagulation factor X (factor Xa), measuring factor Xa activity can determine the presence of rivaroxaban. Results: We report a magnetic biosensing platform capable of measuring the activity of factor Xa using peptide substrates conjugated with magnetic nanoparticles (MNPs). Due to the size of factor Xa, a new method of solution-phase assays was proposed for magnetic biosensing platforms to address issues with immobilized peptides on the sensors. This method was validated with factor Xa and trypsin, both of which are serine proteases. In the solution-phase assays, samples with either the enzymes of interest or no enzyme were simultaneously measured, and the activity of the enzyme was estimated using the difference between the measurements. Unlike conventional optical methods, our platform was capable of detecting the activity of factor Xa at 2 μg mL⁻¹ with a 30 min sample incubation. Significance: The assay using giant magnetoresistive biosensors outperformed conventional optical techniques. This platform can facilitate the determination of the presence of rivaroxaban and assist physicians in deciding on appropriate treatments for patients.