This study introduces a beta-cyclodextrin/reduced graphene oxide/indium tin oxide (β-CD/rGO/ITO) microelectrode platform for the real-time, non-invasive, and label-free detection of N-acetylneuraminic acid (Neu5Ac) on red blood cell (RBC) surfaces. By leveraging advances in CRISPR-Cas9 technology, genetically modified pig-derived RBCs with reduced immunogenicity have been developed for xenotransfusion applications. However, elevated Neu5Ac expression in some modified RBCs complicates immunological assessment. The developed platform demonstrated exceptional sensitivity, specificity, and reusability, enabling selective monitoring of Neu5Ac levels on RBC surfaces through interactions with Siglec-1, a macrophage receptor for Neu5Ac. Results effectively distinguished genetically modified pig RBCs (double knockout (DKO), triple knockout (TKO), and quadruple knockout (QKO)) from human RBCs (HuRBCs) based on Neu5Ac expression. DKO RBCs exhibited the highest impedance value (50.43 ± 3.28 Ω), while HuRBCs showed the lowest (6.29 ± 3.14 Ω). The platform's 98.6% reusability after sialidase treatment and its ability to screen substances that inhibit Neu5Ac-mediated immune recognition highlight its potential for therapeutic applications. These features establish the β-CD/rGO/ITO microelectrode as a valuable tool for quality assessment of xenogeneic RBCs and for advancing clinical and translational applications in xenotransfusion medicine.
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