A simple metal-free electrochemical immunosensor based on nitrogen-doped graphene acid (NGA) for the detection of 25-hydroxy vitamin D3 (25-OH D3) is presented. The use of NGA, with its well-defined chemical composition, high density of carboxyl functional groups, and excellent biocompatibility, provided a stable and efficient platform for biomolecule immobilization without the need for complex surface modifications. The proposed immunosensor demonstrated high sensitivity (1.97 kΩ ng‒1 mL cm‒2) and a working range of 3.96 to 48.83 ng mL‒1, aligning with clinically relevant vitamin D levels. Moreover, the developed immunosensor exhibited excellent specificity, with minimal cross-reactivity and strong resistance to non-specific adsorption. Furthermore, it maintained stable electrochemical performance for up to 30 days, demonstrating its suitability for practical applications. Compared to existing vitamin D sensors, our platform introduces a streamlined fabrication process that avoids heavy metals, improves reproducibility, and enhances accessibility for point-of-care diagnostics.

Department of Biomedical Engineering Faculty of Electrical Engineering and Communication Brno University of Technology Technická 10 Brno 616 00 Czech Republic

Department of Microelectronics Faculty of Electrical Engineering and Communication Brno University of Technology Technická 10 Brno 616 00 Czech Republic

Department of Surgery 1 Faculty of Medicine and Dentistry Palacký University Olomouc and University Hospital Olomouc Olomouc 771 47 Czech Republic

IT4Innovations VSB Technical University of Ostrava 17 Listopadu 2172 15 Ostrava Poruba 708 00 Czech Republic

Nanotechnology Centre Centre for Energy and Environmental Technologies VSB Technical University of Ostrava 17 Listopadu 2172 15 Ostrava Poruba 708 00 Czech Republic

Regional Centre of Advanced Technologies and Materials Palacký University Olomouc Šlechtitelů 27 Olomouc 783 71 Czech Republic

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