A lipoprotein-specific aptamer biosensor for the detection of mycoplasma-contamination in T cell culture

Mycoplasma contamination remains a leading cause of insidious cell culture failure, altering cellular physiology while evading standard antibiotic treatments due to the lack of a cell wall. Current electrochemical detection strategies rely mainly on DNA-based assays that require cell lysis and DNA extraction, thereby increasing assay complexity and time. Here we report an electrochemical aptamer biosensor for the direct detection of Mycoplasma hyorhinis ( MHO ) through specific recognition of its high-affinity transport protein (p37). This strategy enables label-free detection of Mycoplasma and secreted p37 protein with minimal sample processing. The biosensor detected MHO in buffer and in a co-culture medium with human T cells, with a detection limit of ~2 cells/mL, a quantification limit of ~7 cells/mL, and a sensitivity of 16.9 ± 0.7%∆ R ct /cells/mL. Additionally, the biosensor response in MHO sample supernatants was investigated to assess its sensitivity to the MHO lipoproteins (p37) released into the extracellular medium during growth and lysis. The results confirmed that the biosensor response originates from the released p37 protein even in the absence of MHO cells, with a consistently lower response compared to samples containing MHO cells. This study lays the foundation for a non-invasive, electrochemical Mycoplasma monitoring tool to track contamination in human T-cell cultures, supporting quality control in high-value cell therapy manufacturing workflows.